Antivenoms and methods for making antivenoms

ABSTRACT

The production of antivenoms in non-mammals and improvements in the effectiveness of both non-mammalian antivenoms and mammalian antivenoms so that they are more suitable for treatment of humans and animals as well as for analytical use.

DESCRIPTION Background of the Invention

The present invention relates to antivenoms suitable for treatment ofhumans and animals as well as for analytical use.

I. VENOMS

A toxin is a single protein or peptide that has deleterious effects inman or animals. A venom comprises a plurality of toxins; they arerelatively complex mixtures of proteins and peptides that can causeconsiderable morbidity and mortality in humans and animals.

The chemical actions of and biological reactions to venoms are asdiverse as their sources. Depending on the nature of the venoms, theirtoxic effects may be evident in the cardiovascular, hematologic,nervous, and/or respiratory systems.

Each region of the world has its own particularly troublesome venomousspecies. Within Eukaryota (see Table 1), some specific venom sourcesfrom the Animalia kingdom are most notable.

A. EUKARYOTA

i) Chordata. A number of Chordata classes are sources of venoms (e.g.Amphibians, Fish, Reptiles). Among Reptiles, the most significant orderis snakes. Snake venom is a relatively complex mixture of enzymes,non-enzymatic proteins and peptides, and as yet unidentified compounds.W. A. Wingert and J. Wainschel, S. Med. J. 68:1015 (1975). D. C.Christopher and C. B. Rodning, S. Med. J. 79:159 (1986).

                  TABLE 1                                                         ______________________________________                                        Phylogeny of Toxin- and Venom- Producing Organisms                            ______________________________________                                        SUPERKINGDOM:      PROKARYOTA                                                 KINGDOM:           MONERA                                                     DIVISION:          BACTERIA                                                   SUPERKINGDOM:      EUKARYOTA                                                  KINGDOM:           FUNGI                                                      KINGDOM:           PLANTAE                                                    KINGDOM:           ANIMALIA                                                   PHYLUM:            CHORDATA                                                   CLASSES:           Amphibia                                                                      Reptilia                                                                      Pisces                                                     PHYLUM:            ARTHROPODA                                                 CLASSES:           Arachnida                                                                     Insecta                                                                       Myriapoda                                                  PHYLUM:            COELENTERATA                                               PHYLUM:            MOLLUSCA                                                   ______________________________________                                    

While there are some chemical similarities, the venom of each speciesexhibits its own characteristic toxicity. M. J. Ellenhorn and D. G.Barceloux, Medical Toxicology, Ch.39 (Elsevier Press (1988).

Of the over 100 species of snakes in the United States, approximately10% are poisonous. H. M. Parrish, Public Health Rpt. 81:269 (1966). Themajority of these are from the family Crotalidae. The venomous speciesinclude the rattlesnakes (Crotalus), cottonmouths and copperheads(Agkistrodon), and pigmy and massassauga rattlesnakes (Sistrurus). Thereare also poisonous members of the Elapidae family, the coral snakes(Micruroides). F. E. Russell et al., JAMA 233:341 (1975). ii)Arthropoda. In the Arthropoda phylum, an important class is Arachnida.Among Arachnida, scorpions (Order Scorpiones) produce the mostsignificant venoms. While scorpion venoms are also complex mixtures,there has been some success identifying their active agents.Approximately thirty different protein neurotoxins, each having amolecular weight of about 7000 daltons, have been isolated. M. E. Ayeband P. Delori, In: Handbook of Natural Toxins, Vol.2, Insect Poisons,Allergens, and Other Invertebrate Venoms, (Anthony T. Tu, Ed.)(MarcelDekker 1984 , Chapter 18 (pp. 607-638). Of the approximately 650scorpion species, the most dangerous belong to the Buthidae family andthe genuses Tityus (North and South America), Centruroides (U.S. andMexico), Centrurus (Mexico), Androctonus (Mediterranean/North Africa),Buthacus (Mediterranean/North Africa), Leiurus (Mediterranean/ NorthAfrica), Buthotus (Mediterranean/North Africa), Buthus(Mediterranean/North Africa), and Parabuthus (South Africa). F. Hassan,In: Handbook of Natural Toxins, Vol.2, Insect Poisons, Allergens, andOther Invertebrate Venoms, (Anthony T. Tu, Ed.)(Marcel Dekker 1984),Chapter 17 (pp. 577-605). iii) Coelenterata. In the Coelenterata phylum,jelly fish are an important venomous species; the venom from Chironexfleckeri is among the most potent and medically significant. In thewaters off Northern Australia, about one fatality occurs each year. J.Lumley et al., Med. J. Aust. 148, 527 (1988). Several toxic fractionshave been characterized from C. fleckeri venom including two highmolecular weight myotoxins (R. Endean, Toxicon 25, 483 (1987)) andseveral low molecular weight toxins having hemolytic or dermonecroticproperties (C. E. Olson et al., Toxicon 22, 733 (1984); E. H. Baxter andA. G. M. Marr, Toxicon 7, 195 (1969)). iv) Mollusca. In the Molluscaphylum, the most significant venomous members are the coneshells(Conidae) which produce potent myotoxins that can be fatal. G. G.Habermehl, Venomous Animals and Their Toxins (Springer-Verlag, Berlin1981). Little is known about the structure of the molluscan myotoxins.

B. PROKARYOTA

Prokaryotes are an important source of toxins. Most bacterial toxins,for example, are well known. Among species of bacteria, the mostnotorious toxin sources are certainly Clostridum botulinum andClostridium parabotulinum. The species produce the neurogenic toxinknown as botulinus toxin. While a relatively rare occurrence in theUnited States, involving only 355 cases between 1976 and 1984 (K. L.MacDonald et al., Am J. Epidemiology 124, 794 (1986)), the death ratedue to the botulism toxin is 12% and can be higher in particular riskgroups. C. O. Tacket et al., Am. J. Med. 76, 794 (1984).

Many other bacteria produce protein toxins of significance to humans,including Bacillus anthracis, Bordetella pertussis (diptheria),Pasteurella pestis, Pseudomonas aeruginosa, Streptococcos pyrogenes,Bacillus cereus, E. coli, Shigella, Staphylococcus aureus, Vibriocholerae, and Clostridium tetani. Thorne and Gorbach, Pharmacology ofBacterial Toxins, In: International Encyclopedia of Pharmacology andTherapeutics, F. Dorner and J. Drews (eds.), Pergamon Press, Oxford(1986), pp. 5-16.

II. TREATMENT

As noted above, a toxin is defined as a single protein or peptide and avenom is defined as comprising a plurality of toxins. Both toxin andvenom have been used as antigen for treatment.

Exposure to most venoms in humans does not result in protectiveimmunity. Furthermore, all attempts to create protective immunityagainst venoms with vaccines have failed. F. E. Russell, JAMA 215:1994(1971) (rattlesnake venom). By contrast, there has been success creatingprotective immunity against individual toxins, including diptheria (F.Audibert et al., Proc. Natl. Acad. Sci USA 79:5042 (1982)) and tetanusvaccines. J. E. Alouf, Ann Inst. Pasteur/Microbiol. 136B, 309 (1985).

A. ACTIVE IMMUNIZATION

Tetanus toxoid injections provide an effective protection because theyelicit a low level of circulating antibody and establish immunologicalmemory. When exposed to a low dose of the tetanus organism and toxin,the immunized animal can neutralize the organism and toxin before theinfection develops.

In the case of animal venoms, such prophylactic measures have not beenfeasible. First, many animal venoms are too difficult or too expensiveto obtain to immunize a population where a relatively small percentageof that population will be exposed to the animal venom. Second, even ifthey can be obtained, animal venoms, unless detoxified, may cause moremorbidity when administered to a large population than would be causedby the venomous animals themselves. Third, even if the venom isaffordable, obtained in sufficient quantity, and detoxified, it isextremely difficult to achieve the titer of circulating antibodynecessary to neutralize the infusion of what can be a large amount ofvenom (up to one gram of animal venom as compared with nanogram orpicogram amounts of tetanus toxin). Finally, even with successfulimmunization, immunological memory is too slow to respond to theimmediate crisis of envenomation.

Although active immunization with venoms has the above-named problems,some investigators have chosen to pursue research in this area ratherthan in the area of passive immunization, arguing that passiveimmunization is too long and expensive. These investigators have madesome progress in the method of immunization by using liposomes. R. R. C.New et al., New Eng. J. Med. 311 56 (1984). T. V. Freitas et al.,Toxicon 27:341 (1989).

B. PASSIVE IMMUNIZATION

Because the problems with active immunization have not been overcome,the only treatment available for venoms is passive immunization. Passiveimmunization, like active immunization, relies on antibodies binding toantigens. For our purposes here, antitoxin refers to antibody raisedagainst a single toxin. Antivenom refers to antibody raised againstwhole venom.

In the case of passive immunization, the antibody used to bind the venom(antigen) is not made in the animal afflicted with the venom. Generally,an immune response is generated in a first animal. The serum of thefirst animal is then administered to the afflicted animal (the "host")to supply a source of specific and reactive antibody. The administeredantibody functions to some extent as though it were endogenous antibody,binding the venom toxins and reducing their toxicity. (It is not knownwhether the antibody directly blocks the action of venom toxins ormerely carries venom toxins out of the blood stream.)

i. Raising Antivenoms. The first step in treatment by passiveimmunization involves raising an antibody with reactivity that isspecific for the venom. Such an antibody is referred to as an antivenom.As noted above, venoms pose unique problems for immunization. They areoften expensive and available in only small amounts. Furthermore,because they are toxic, they can do great damage before, and in somecases without, generating an immune response.

Usually the problem of a toxicity is approached by modifying the venomin some manner. Modification of venoms, however, creates new problems.On the one hand, the modification may have so damaged the venom that itis largely non-immunogenic. On the other hand, while not renderednon-immunogenic, the modification may have so altered the venom that anew antigenicity is created. That is, antibody raised to the modifiedvenom is directed to the modification as part of the antigenic site. Inthis case, the antibody raised to the modified venom may not react withthe unmodified venom (as it will be found in its natural state).Finally, the modification may itself be toxic or cause unexpected sideeffects.

Immunization with venoms is also complicated by their complexcomposition. Venoms are remarkably heterogeneous. Furthermore, thevarious components of venoms are present in different amounts. There issome concern that immunization with whole venom will not result inantibody reactive with all venom components.

ii. Administration. The second step in treatment by passive immunization(assuming, of course, the problems with the first step have been dealtwith), involves the administering of antivenom to the host. The firstconcern is whether the host will tolerate the administration of"foreign" antibody. In other words, will the host's immune systemrecognize the administered antibody as antigen and mount an adverseresponse?

Adverse host responses are typically of two types, immediate anddelayed. Immediate reactions are also of two types: 1) anaphylaxis, and2) Arthus reaction. Anaphylaxis is IgE mediated and requiressensitization to antigen. The Arthus reaction is complement dependentand requires only antibody-antigen complexes. Both immediate types ofreactions are referred to as hypersensitivity reactions; the hostresponds as if primed by a first exposure. Such immediate reactions canbe acute. Indeed, anaphylaxis, if untreated, can lead to respiratoryfailure and death.

Delayed reactions are caused by a host primary immune response to theforeign proteins of the antivenom. The reaction, called "serumsickness," is characterized by fever, enlarged lymph glands, and jointpain. These symptoms are apparent a number of days after passiveimmunization and gradually subside.

The next concern about administering antivenoms is the dose. Withoutknowing the amount of venom in the host it is difficult to know theamount of antivenom needed to treat the host. Furthermore, even if theamount of venom can be estimated, how is the amount of antivenom to bemeasured? Some approaches measure antivenom in units of volume. Such anapproach does not account for different antivenom antibodyconcentrations within the same volume of serum.

iii. Commercial Antivenoms. Antivenoms have been raised in a number ofmammals. See J. C. Perez et al., Toxicon 22:967 (1984) (mice). D. Iddonet al., Toxicon 26:167 (1988) (mice). R. A. Martinez et al., Toxicon27:239 (1989) (mice). M. E. Ayeb and P. Delori, In: Handbook of NaturalToxins, Vol.2, Insect Poisons, Allergens, and Other Invertebrate Venoms,(Anthony T. Tu, Ed.) (Marcel Dekker 1984), Chapter 18 (pp. 607-638)(rabbits). F. E. Russell et al., Toxicon 8:63 (1970) (goats). S. C.Curry et al., J. Toxico).--Clin. Toxicol. 21417 (1983-1984) (goats). F.Hassan, In: Handbook of Natural Toxins, Vol. 2, Insect Poisons,Allergens, and Other Invertebrate Venoms, (Anthony T. Tu, Ed.) (MarcelDekker 1984), Chapter 17 (pp. 577-605) (cows). Horses, however, are theanimal of choice by an overwhelming number of investigators andcommercial antivenom producers. World Health Organization PublicationNo. 58 (Geneva 1981).

Horses are sturdy and tolerant to the antibody-raising process. Mostimportantly, they yield large volumes of blood (as much as ten litersper bleeding for large animals).

There are significant disadvantages, however, when using horses forantivenom production. First, for large production of antivenoms, horsesmore than 5 years old and usually less than 8 years old are required.Second, because new horses are easily killed or injured, productionshould be under veterinary care and supervision. Third, tetanus is knownto be a common disease among horses; animals must be immunized as soonas they are introduced to the farm. F. Hassan, In: Handbook of NaturalToxins, Vol. 2, Insect Poisons, Allergens, and Other InvertebrateVenoms, (Anthony T. Tu, Ed.) (Marcel Dekker 1984), Chapter 17 (pp.577-605). Fourth, large amounts of venom (antigen) are required forimmunization in order to generate a satisfactory immune response inhorses. Fifth, horse antibody binds and activates human and othermammalian complement pathways, leading (at the very least) to complementdepletion and (at worst) to a more acute reaction by the host. Mostcommercial antivenoms contain anticomplementary activity. S. K.Sutherland, Med J. Australia 1: 613 (1977). Sixth, some humans arehypersensitive to horse serum proteins and may react acutely to evenvery small amounts of horse protein. P. A. Christensen, In: Snake Venoms(Springer-Verlag 1979), Chapter 20 (pp. 825-846).

In spite of these problems, horse antivenom is the only specifictreatment of most venom poisonings known at the present time. It isconsidered vital for treating severe cases of snake envenomation. H. M.Parrish and R. H. Hayes, Clin. Tox. 3:501 (1970). Similarly, horse serumcontaining antivenoms is considered life-saving in the treatment ofscorpion stings. F. Hassan, In: Handbook of Natural Toxins, Vol. 2,Insect Poisons, Allergens, and Other Invertebrate Venoms, (Anthony T.Tu, Ed.) (Marcel Dekker 1984), Chapter 17 (pp. 577-605).

In the United States, the primary commercial producer of antivenom tosnake venoms is Wyeth Laboratories (Marietta, Pennsylvania). To make auseful antivenom to members of the Crotalidae family, horses areimmunized with a mixture of venom from four distinct species. To reducetheir toxicity, the venoms are modified by treatment with formalin. Toprolong their absorption, the modified venoms are mixed with aluminumhydroxide gel. H. M. Parrish and R. H. Hayes, Clin. Tox. 3:501 (1970).Serum is collected and total antibody is precipitated. During thecollection process, it is reported that the ammonium sulfateprecipitation destroys up to one half of the neutralizing antibodies ofthe crude antivenom. M. J. Ellenhorn and D. G. Barceloux, MedicalToxicology, Ch.39 (Elsevier Press 1988).

One of the most difficult aspects of clinical management of envenomationis the lack of standardization of antivenoms. The recommended dosages oftherapeutic horse-derived antivenoms is usually given in units ofvolume. For example, treatment with the Wyeth antivenom is measured interms of vials of antivenom; each vial represents approximately 10 mlsof antivenom in solution. D. C. Christopher and C. B. Rodning, S. Med.J. 79:159 (1986). M. J. Ellenhorn and D. G. Barceloux, MedicalToxicology, Ch.39 (Elsevier Press 1988). H. M. Parrish and R. H. Hayes,Clin. Tox. 3:501 (1970). F. E. Russell et al., JAMA 233:341 (1975).

The potency of individual lots of antivenoms will vary because of twoprincipal factors. First, because whole antisera or immunoglobulinfractions are used and the specific antibody titer per unit volume willvary from animal to animal and from day to day, the amount ofvenom-reactive antibodies will differ from preparation to preparation.Second, refinement procedures such as ammonium sulfate precipitation andpepsin digestion can reduce the yield of active antibody, causingvariations in the titer of active ingredient per unit volume. Thesedifficulties are exacerbated when antivenom is raised against a set ofvenoms in order to treat a range of species. That is, when certainspecies are more diverged from the immunizing group, it is moredifficult to determine how much antivenom will be required.

Because of the array of common and serious side effects of unpurifiedantivenoms the physician must exercise caution not to give excessiveamounts of horse product. Patients who receive seven or more vials ofthe Wyeth preparation are reported to invariably develop serum sickness;approximately 80% of patients overall who receive the preparationdevelop serum sickness within three weeks. M. J. Ellenhorn and D. G.Barceloux, Medical Toxicology, Ch.39 (Elsevier Press 1988).

iv. Avoidino Side Effects. Because the commercial antivenoms presentlyavailable can cause their own adverse reactions, the risk of possibledeath or serious injury from the venom must be weighed against the riskof a hypersensitivity reaction to horse serum. Before administration ofhorse serum, good medical practice requires that serum sensitivity testsbe performed. H. M. Parrish and R. H. Hayes, Clin. Tox. 3:501 (1970).

Serum sensitivity is typically performed by subcutaneously injecting asmall amount of diluted serum in the arm of the patient. A salt solutionis injected in the other arm as a control. Normally, a positivehypersensitivity test is indicated by no more than formation of a welton the skin surface with surrounding swelling. Some patients, however,develop anaphylactic shock, i.e. a full hypersensitivity reaction. It isrecommended in the medical literature that adrenalin be available forthese cases.

While sensitivity testing has its advantages, it is generallyacknowledged that it has no predictive value for serum sickness andreactions due to complement activation. World Health OrganizationPublication No. 58 (Geneva 1981). Thus, all patients must be regarded aspotential "reactors" and all drugs and equipment required for dealingwith reactions must be available before antivenoms are administered.

V. PURIFICATION

One approach to avoiding side effects deserves special note. It has beentheorized that the high incidence of side effects with currentcommercial horse antivenoms is due to the bulk of irrelevant protein inthese preparations. (Protein other than specific antibody is consideredto be irrelevant protein.) Under this theory, the removal of irrelevantprotein would reduce the burden of foreign protein and, thereby, reducethe incidence of adverse immune responses.

F. Hassan, In: Handbook of Natural Toxins, Vol. 2, Insect Poisons,Allergens, and Other Invertebrate Venoms, (Anthony T. Tu, Ed.) (MarcelDekker 1984), Chapter 17 (pp. 577-605) attempted a crude purification ofhorse antivenom. First, the horse serum was subjected to a mild pepsindigestion followed by ammonium sulfate precipitation. Then, theprecipitate was heat denatured; the heat-labile fraction was removed.Unfortunately, approximately one-third of the initial antivenom activitywas reported to be lost by this method.

A handful of antivenom investigators have considered immunoaffinitypurification. However, most studies have only examined antibodies to asingle toxin. C. C. Yang et al., Toxicon 15, 51 (1977) attemptedimmunoaffinity purification of antibody to a toxin in a snake venom.These investigators used cobrotoxin, a neurotoxic crystalline proteinisolated from the venom of Taiwan cobra (Naja naja atra); whole venomwas not used. Cobratoxin attached to Sepharose (CNBr-activated Sepharose4B) was used as an antigen matrix and formic acid was used to elute thetoxin-specific antibodies. The immunoaffinity purified neutralizingcapability than the unpurified antiserum.

V. Kukongviriyapan et al., J. Immunol. Meth. 49:97 (1982) followed witha similar purification scheme. Again, whole venom was not used. Theseinvestigators used Naja naja siamensis toxin 3, purified according tothe method of E. Karlsson et al., Eur. J. Biochem. 21, 1 (1971). Anumber of antigen matrices were studied, including toxin-Sepharose(CNBr-activated Sepharose 4B), toxin-succinylaminoethyl Sepharose,toxin-albumin Sepharose, and toxin-succinylaminoethyl Biogel. Horseantibody was used. Unfortunately, only approximately 5% of the appliedprotein was reportedly bound and the destruction of antigenic sites onthe immobilized toxin occurred extensively. Most importantly, thetoxin-neutralizing capacity recovered in the purified antibodyrepresented only approximately one-third that of the unpurifiedglobulin.

M. E. Ayeb and P. Delori, In: Handbook of Natural Toxins, Vol. 2, InsectPoisons, Allergens, and Other Invertebrate Venoms, (Anthony T. Tu, Ed.)(Marcel Dekker 1984), Chapter 18 (pp. 607-638) also followed the Yang etal. procedure and applied it to purifying antibodies against individualscorpion neurotoxins. Again, whole venom was not used. Theseinvestigators used toxin II of A. australis Hector. While theseinvestigators did not report yields, they noted that formic acid causeddenaturation of the antibody.

B. Lomonte et al., Toxicon 23:807 (1985), purified antibodies against B.Asper myotoxin coupled to CNBr-activated Sepharose 4B. The antimyotoxinwas only 0.5-1.0% of the antivenom protein and was found to be lesseffective than crude antivenom in neutralizing the lethal effects of thevenom.

J. B. Sullivan's research group examined immunoaffinity purificationwith whole venoms. See J. B. Sullivan et al., J. Vet. Hum. Toxicol.24:192 (Suppl.) (1982). J. B. Sullivan and F. E. Russell, Proc. WesternPharmacol. Soc. 25:185 I982 . J. B. Sullivan and F. E. Russell, ToxiconSuppl. 3:429 (1983). W. S. Jeter et al., Toxicon 21:729 (1983). D.Bar-Or et al, Clin. Tox. 22:1 (1984). F. E. Russell et al., Am. J. Trop.Med. Hyg. 34:141 (1985). J. B. Sullivan, Ann. Emerg. Med. 16:938 (1987).All of this work was performed with a polyacrylamide resin and trappingas the means for associating the venom with the resin.

Trapping involves suspending molecules in a gel. Trapping does notinvolve attachment (covalent or non-covalent) of the venom via areactive group on the resin; without such an attachment, venom can findits way through the matrix and end up in the eluate. Furthermore, asvenom from the antigen matrix finds its way out of the suspension, thereis a progressive reduction in the antibody binding capacity of theantigen matrix. Loss of binding capacity renders the matrixnon-recyclable, i.e. one cannot recover the same amount of purifiedantibody in subsequent purifications.

Polyacrylamide has several drawbacks. First, polyacrylamide has lowporosity and, hence, can sterically hinder some antibody-antigeninteractions, thereby reducing the antibody binding capacity of thepolyacrylamide-antigen matrix. A. Johnstone and R. Thorpe,Immunochemistry in Practice, 2d Edition (Blackwell ScientificPublications 1987), p. 209. Second, polyacrylamide itself is aneurotoxin; there is a concern that polyacrylamide may leech from thepolyacrylamideantigen matrix into the eluate and contaminate purifiedantibody.

vi. Non-mammalian Sources of Antivenoms. As mentioned above, mostantivenoms are made in mammals and the overwhelming majority have beenmade in horses. There have been only a few attempts made at raisingantivenoms in non-mammals. A. Polson et al Immunol. Comm. 9:495 (1980),attempted to raise antivenoms against snake venoms in chickens. Theirwork was unsuccessful; the chicken immunoglobulin showed no protectiveactivity against the venom in an assay performed in mice. It wasspeculated that chicken antibody interactions with venom are inherentlyweaker and less stable than those of horse antibody.

SUMMARY OF THE INVENTION

The present invention relates to antivenoms suitable for treatment ofhumans and animals as well as for analytical use.

In one embodiment, the present invention contemplates a compositioncomprising polyvalent antivenom, comprised of immunoglobulin of whichgreater than fifty percent is venom-reactive, and having two or moremonovalent subpopulations. The composition is preferably in an aqueoussolution in therapeutic amounts and intravenously injectable. Thepolyvalent antivenom preferably has reactivity to C. atrox, B. atrox. C.adamanteus and C. durissus terrificus venom. Preferably, one of themonovalent subpopulations comprises antibody with reactivity to C.durissus terrificus venom. The polyvalent antivenom preferably compriseshorse antibody.

In another embodiment, the present invention contemplates a compositioncomprising polyvalent antivenom, comprised of immunoglobulin of whichgreater than fifty percent is venom-reactive, and having two or moremonovalent subpopulations, wherein the polyvalent antivenom is derivedfrom a first polyvalent antivenom comprised of immunoglobulin of whichless than fifty percent is venom-reactive, and has substantially thesame spectrum of reactivity as said first polyvalent antivenom.

In still another embodiment, the present invention contemplates acomposition comprising, polyvalent antivenom, derived from a firstpolyvalent antivenom comprised of immunoglobulin of which less thanfifty percent is venom-reactive, and having substantially the samespectrum of reactivity as the first polyvalent antivenom. Thecomposition is preferably in an aqueous solution in therapeutic amountsand intravenously injectable. The polyvalent antivenom preferably hasreactivity to C. atrox. B. atrox, C. adamanteus and C. durissusterrificus venom. The polyvalent antivenom preferably comprises horseantibody. The composition preferrably comprises polyvalent antivenomcomprising two or more monovalent subpopulations.

In a preferred embodiment, the present invention contemplates acomposition comprising venom-neutralizing avian antivenom. Thecomposition is preferably in an aqueous solution in therapeutic amountsand intravenously injectable. Preferrably, the avian antivenom ischicken antivenom and the chicken antivenom is comprised of yolkimmunoglobulin. It is desirable that the avian antivenom is comprised ofprotein comprised of greater than 90% immunoglobulin and greater than50% venom-reactive immunoglobulin. Preferably, the avian antivenom iscomprised of protein comprised of greater than 90% immunoglobulin andgreater than 99% venom-reactive immunoglobulin. It is desirable that theavian antivenom is polyvalent. Preferably, the avian antivenom is highavidity chicken antivenom.

The present invention also contemplates an antigen matrix useful forpurification of antivenom. In one embodiment, the antigen matrixcomprises C. durissus terrificus venom attached to an insoluble support.In one embodiment, the antigen matrix comprises a plurality of venomsattached to an insoluble support. Preferably, the attachment is covalentattachment. It is desirable that the insoluble support prior toattachment to the plurality of venoms comprise a resin having aldehydegroups. Preferably, the plurality of venoms comprises C. atrox. B.atrox, C. adamanteus and C. durissus terrificus venom.

The present invention also contemplates a method for immobilizing wholevenom. In one embodiment, the method comprises a) providing an insolublesupport; b) providing two or more whole venoms; and c) attaching two ormore whole venoms to the insoluble support. Preferably, the attaching iscovalent attaching. It is desirable that the insoluble support comprisesa resin comprising aldehyde-activated agarose.

In another embodiment, the method for immobilizing whole venom comprisesa) providing an insoluble support; b) providing a single whole venom;and c) attaching the single whole venom to the insoluble support bycovalent binding.

The present invention also contemplates a method of producing antivenom.In one embodiment, the producing method comprises a) providing one ormore immunizing venoms; b) providing at least one avian species; and c)immunizing the avian species with one or more immunizing venoms, so thata neutralizing antivenom is produced. Preferably, the avian speciescomprises chickens.

The present invention also contemplates a method for purifyingantivenom. In one embodiment, the purifying method comprises a)providing, in any order: i) at least one antivenom comprisingimmunoglobulin of which less than 50% is venom-reactive, ii) at leastone antigen matrix comprising at least one venom immobilized on aninsoluble support, iii) at least one first and one second eluent; b)applying antivenom to the antigen matrix so that greater than ninetypercent of the venom-reactive immunoglobulin binds the venom immobilizedon the insoluble support of the antigen matrix; c) dissociating at leastfifty percent of the bound venom-reactive immunoglobulin from the venomwith the first eluent; and d) stripping the antigen matrix ofsubstantially all of the venom-reactive immunoglobulin with the secondeluent so that the antigen matrix is recyclable. It is desirable thatthe antivenom comprises horse antivenom. Preferably, the antivenomcomprises chicken antivenom and the chicken antivenom comprises yolkimmunoglobulin.

The present invention also contemplates a method of analyzing antivenom.In one embodiment, the analyzing method comprises a) providing, in anyorder, i) a first and a second immunizing venoms, ii) a solutioncomprising antivenom comprising immunoglobulin subpopulations havingreactivity with the immunizing venoms, iii) a first antigen matrixcomprised of the first immunizing venom immobilized on an insolublesupport, iv) a second antigen matrix comprised of a second immunizingvenom immobilized on an insoluble support, and v) one or more eluents;b) applying the solution of antivenom to the first antigen matrix sothat greater than 90% of the immunoglobulin reactive with the firstimmunizing venom is bound to the first antigen matrix and so that thesolution passes through the first antigen matrix to create a firstflow-through; c) applying the eluent to the first antigen matrix so thatat least fifty percent of the bound immunoglobulin reactive with thefirst immunizing venom is dissociated to create a first eluate; and d)applying, in any order, i) the first flow-through to the second antigenmatrix, followed by the eluent to create a second eluate, ii) the firsteluate to the second antigen matrix so that the solution passes throughthe second antigen matrix to create a second flow-through, followed bythe eluent to create a third eluate. In one embodiment, the methodfurther comprises, after step d), comparing the venom-reactivity of thefirst flow-through, the first eluate, the second flow-through, thesecond eluate, and the third eluate, with the venom reactivity of theantivenom. It is desirable that the antivenom comprises horse antibody.Preferably, the antivenom comprises chicken antibody and the chickenantibody comprises yolk immunoglobulin. It is desirable that, afterapplying the eluent, the first and second antigen matrices are renderedrecyclable. A desirable recycling eluent is guanidine.

The present invention also contemplates a method of treatment. In oneembodiment, the treatment comprises: a) providing i) avian antivenom inan aqueous solution in therapeutic amounts that is intravenouslyinjectable, ii) at least one envenomed subject b) intravenouslyinjecting the avian antivenom into the subject. Preferably, the avianantivenom is chicken antivenom and the chicken antivenom comprises yolkimmunoglobulin. It is desirable that the avian antivenom comprisesimmunoglobulin of which greater than fifty percent is venom-reactive.Preferably, the subject is a mammal.

In an alternative embodiment, the present invention contemplates amethod of treatment, comprising: a) providing i) polyvalent antivenom inan aqueous solution in therapeutic amounts that is intravenouslyinjectable, comprising immunoglobulin of which greater than fiftypercent is venom-reactive, and having two or more monovalentsubpopulations, ii) at least one envenomed subject; b) intravenouslyinjecting the polyvalent antivenom into the subject. It is desirablethat the polyvalent antivenom is horse antivenom.

It is not intended that the present invention be limited by the sourceof the venom used for immunizing, purifying or analyzing. Similarly, itis not intended that the present invention be limited by the source ofthe venom for which the antivenom compositions of the present inventionare reactive. For example, the present invention contemplates venomsselected from the group consisting of Chordata, Arthropoda andCoelenterata venoms. Venoms selected from the group consisting of snakevenoms, spider venoms, scorpion venoms or jelly fish venoms arespecifically contemplated. The present invention also contemplates venomselected from the group consisting of Crotalus scutulatus, Notechisscutatus, Acanthophis antarcticus, Oxyuranus scutellatus, Pseudonajatextilis, Pseudechis australis, Enhydrina schistosa, Ophiophaous hannah,Vipera ammodytes, Vipera aspis, Vipera berus, Vipera xanthinapalestinae, Vipera lebetina, Cerastes cerastes. Cerastes vipera, Bitisarietans, Bitis gabonica, Vipera russelli, Echis carinatus, Trimeresurusflavoviridis, Agkistrodon halys, A. piscivorus, A. contortrix, Najanaja, Naja n. haje, Naja n. kaouthia, Naja n. oxiana, Naja n. sputatrix,Naja n. atra, Naja nivea, Naja nigrocollis, Hemachatus hemachatus,Dendroaspis angusticeos, Dendroaspis jamesonii, Dendroaspis polylepis,Dendroaspis viridis, Bungarus caerulus, Bungarus fasciatus, Bungarusmulticinctus, Agkistrodon rhodostoma, Agkistrodon acutus, Bothropsatrox, Bothrops jararaca, Bothrops jararacussu, Bothrops alternatus,Lachesis muta, Micrurus corralus, Micrurus fulvius, Micrurus frontalis,Micrurus niagrocinctus, Leiurus quinouestriatus, Tityus serrulatus,Centruroides suffusus, Centruroides noxius, Centruroides sculpturatus,Androctonus australis, Buthotus judaicus, Buthus tamalus, Latrodectusmactans, Latrodectus hesperus, Loxosceles reclusa, and Chironex fleckerivenom. Preferably, the antivenoms of the present invention react with C.atrox, B. atrox, C. adamanteus and C. durissus terrificus venoms.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic, showing a preferred embodiment of the method ofthe present invention.

FIG. 2 is a schematic, showing one approach to venom epitopedeterminations of the present invention.

FIG. 3 is a schematic, showing a preferred approach to venom epitopedeterminations of the present invention.

FIG. 4 is a schematic, showing a preferred approach to antivenomimmunoaffinity purification of the present invention.

FIG. 5 is a Western Blot, showing the reactivity of antivenoms raisedagainst modified venoms.

FIG. 6 shows the reactivity by ELISA of antivenom raised in differentchickens against snake venom.

FIG. 7 shows the reactivity by ELISA of two preparations of antivenomraised in one chicken over three hundred days apart.

FIG. 8 shows the increase in elution efficiency observed with increasedresidence time of a non-denaturing eluent.

FIG. 9 shows an immunoaffinity purification profile for a preferredembodiment of the method of the present invention.

FIG. 10 shows SDS-PAGE analysis of chicken and horse antivenoms.

FIGS. 11(A)-11(C) show the reactivity by ELISA of mammalian antivenompurified using one embodiment of the method of the present invention.

FIG. 12 shows the reactivity by ELISA of avian antivenom purified usingone embodiment of the method of the present invention.

FIG. 13 compares the reactivity by ELISA of two antivenom preparations,purified using two different embodiments of the method of the presentinvention.

FIG. 14 compares the reactivity by ELISA of two antivenom preparations,purified using two different embodiments of the method of the presentinvention.

FIGS. 15(A)-15(C) show the spectrum of reactivity by Western Blot ofantivenoms before and after immunoaffinity purification.

FIG. 16 shows the spectrum of reactivity by Western Blot ofimmunoaffinity purified antivenoms with immunizing and non-immunizingvenoms.

DESCRIPTION OF THE INVENTION

The present invention is directed to antivenoms and method for makingantivenoms. The properties of antivenom of the present invention makethe antivenoms multi-purpose; antivenoms prepared according to thepresent invention are useful for analytical studies in vitro and usefulas therapeutic agents.

The present invention contemplates I) producing antivenoms innon-mammals, and II) increasing the effectiveness of both non-mammalianantivenoms and mammalian antivenoms, however they might have beenproduced. The present invention further contemplates III) treatinghumans and animals by in vivo administration of antivenoms. A preferredembodiment of the method of the present invention is shown in FIG. 1illustrating the temporal relationship of the method steps. Theindividual steps are described separately below.

I. Obtaining Antivenoms in Non-Mammals

A preferred embodiment of the method of the present invention forobtaining antivenoms involves immunization. However, it is alsocontemplated that antivenoms could be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins and/or venoms as well as non-mammals that have antibodies totoxins and/or venoms by virtue of reactions with the administered(non-venom) antigen. An example of the latter involves immunization withsynthetic peptides or recombinant proteins sharing epitopes with venomcomponents.

In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with whole venom(s). It is notintended that the present invention be limited to any particular venom.Venom from all venomous sources (see Table 1) are contemplated asimmunogens.

When immunization is used, the preferred non-mammal is from the classAves. All birds are comtemplated (e.g. duck, ostrich, emu, turkey,etc.). A preferred bird is a chicken. Importantly, chicken antibody doesnot fix mammalian complement. See H. N. Benson et al., J. Immunol.87:610 (1961). Thus, chicken antibody will normally not cause acomplement dependent reaction. A. A. Benedict and K. Yamaga, In:Comparative Immunology (J. J. Marchaloni, Ed.), Ch. 13, Immunoglobulinsand Antibody Production in Avian Species (pp.335-375) (Blackwell, Oxford1966). Thus, the preferred antivenoms of the present invention will notexhibit complement-related side effects observed with antivenoms knownpresently.

When birds are used, it is contemplated that the antivenom will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antivenom from the egg. Laying hens exportimmunoglobulin to the egg yolk ("IgY") in concentrations equal to orexceeding that found in serum. See R. Patterson et al., J. Immunol.89:272 (1962). S. B. Carroll and B. D. Stollar, J. Biol. Chem. 258:24(1983). In addition, the large volume of egg yolk produced vastlyexceeds the volume of serum that can be safely obtained from the birdover any given time period. Finally, the antibody from eggs is purer andmore homogeneous; there is far less non-immunogobulin protein (ascompared to serum) and only one class of immunoglobulin is transportedto the yolk.

It has been noted above that, when considering immunization with venoms,one may consider modification of the venom to reduce its toxicity. Inthis regard, it is not intended that the present invention be limited byimmunization with modified venom. Unmodified ("native") venom is alsocontemplated as an immunogen.

It is also not intended that the present invention be limited by thetype of modification--if modification is used. The present inventioncontemplates all types of venom modification, including chemical andheat treatment of the venom. The preferred modification, however, isheat-inactivation.

It is not intended that the present invention be limited to a particularmode of immunization; the present invention contemplates all modes ofimmunization, including subcutaneous, intramuscular, intraperitoneal,and intravascular injection.

The present invention further contemplates immunization with or withoutadjuvant. (Adjuvant is defined as a substance known to increase theimmune response to other antigens when administered with otherantigens.) If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant--or that thesame adjuvant, once used, be used all the time. While the presentinvention contemplates all types of adjuvant, whether used separately orin combinations, the preferred use of adjuvant is the use of CompleteFreund's Adjuvant followed sometime later with Incomplete Freund'sAdjuvant.

When immunization is used, the present invention contemplates a widevariety of immunization schedules. In one embodiment, a chicken isadministered venom(s) on day zero and subsequently receives venom(s) inintervals thereafter. It is not intended that the present invention belimited by the particular invervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. However, a preferred schedule forimmunization of the present invention is the administration of acocktail of (heat-inactivated) venoms on day zero at 1 mg/ml for eachvenom, with subsequent administrations of the same cocktail(heat-inactivated or native) at the same dose on days 14 and 21, andwith gradually increasing doses ("boosts") up to 10 mg/ml (native) atapproximately two week intervals up to approximately one hundred days.The preferred collection time is sometime after day 100. This preferredimmunization schedule results in the production of high quantities ofreactive chicken antibody (i.e. reactive with the components of theimmunized venom(s)) per ml of egg yolk (i.e. "high titers").Furthermore, this preferred immunization schedule results in theproduction of antivenoms with "high avidity." High avidity is defined asantibody reactivity with multiple epitopes on individual venomcomponents as measured by the formation of precipitin lines inOuchterlony immunodiffusion gels at salt concentrations less than 1.5MNaCl. Antivenoms requiring salt concentrations of 1.5M NaCl or greaterto form precipitin lines are "low avidity" antivenoms. While not limitedto any precise mechanism, high avidity antivenoms have a greaterprobability of neutralizing venom components in vivo.

Where birds are used and collection of antivenom is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that storage of the eggs be performed at 4° C.for less than one year.

It is contemplated that chicken antibody produced in this manner can bebuffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g. immunoaffinity purification).

II. INCREASING THE EFFECTIVENESS OF ANTIVENOMS

When purification is used, the present invention contemplates purifyingto increase the effectiveness of both non-mammalian antivenoms andmammalian antivenoms. Specifically, the present invention contemplatesincreasing the percent of venom-reactive immunoglobulin. When evaluatedfor immunoglobulin content, purity and reactivity, at different stagesof purification, preferred antivenoms of the present invention have thefollowing relationship: less than 50% of the immunoglobulin of thepolyvalent antivenom prior to purification (i.e. of the "firstpolyvalent antivenom") is venom-reactive; greater than 50% of theimmunoglobulin of the polyvalent antivenom after purification isvenom-reactive.

While all types of purification (e.g. purification based on size,charge, solubility, etc.) may be used, the preferred purificationapproach for mammalian antibody is immunoaffinity purification. Thepreferred purification approaches for avian antibody are: A)Polyethylene Glycol (PEG) separation, and B) Immunoaffinitypurification.

A. PEG PURIFICATION

The present invention contemplates that avian antivenom be initiallypurified using simple, inexpensive procedures. In one embodiment,chicken antibody from eggs is purified by extraction and precipitationwith polyethylene glycol (PEG). PEG purification exploits thedifferential solubility of lipids (which are abundant in egg yolks) andyolk proteins in high concentrations of polyethylene glycol 8000. Polsonet al., Immunol. Comm. 9:495 (1980). The technique is rapid, simple, andrelatively inexpensive and yields an immunoglobulin fraction that issignificantly purer in terms of contaminating non-immunoglobulinproteins than the comparable ammonium sulfate fractions of mammaliansera and horse antivenoms. Indeed, PEG-purified antibody is sufficientlypure that the present invention contemplates the use of PEG-purifiedantivenoms in the passive immunization of envenomed humans and animals.

B. IMMUNOAFFINITY PURIFICATION

As noted, immunoaffinity purification is the preferred purificationapproach for both mammalian and avian antivenom. Immunoaffinitypurification is separation based on the affinity of antibody forspecific antigen(s); antibody that binds to specific antigen(s) isseparated from antibody that does not bind (under the conditions used).The present invention contemplates the use of immunoaffinitypurification to dramatically reduce the foreign protein burden ofantivenoms by elimination of irrelevant protein (non-immunoglobulin andnon-antigen-binding immunoglobulin) when the antivenom is usedtherapeutically. While not limited to any specific theory, it iscontemplated that a reduction in the protein burden will be accompaniedby a reduction in side effects associated with passive immunization offoreign protein.

The present invention contemplates immunoaffinity purification by use ofan "antigen matrix" comprised of venom(s) attached to an insolublesupport. Antibody to be purified is applied in solution to the antigenmatrix. The solution passes through the antigen matrix and comprises the"flow through." Antibody that does not bind, if present, passes with thesolution through the antigen matrix into the flow through. To eliminateall non-binding antibody, the matrix is "washed" with one or more washsolutions which, after passing through the matrix, comprise one or more"effluents." "Eluent" is a chemical solution capable of dissociatingantibody bound to the antigen matrix (if any) that passes through theantigen matrix and comprises an "eluate." Antibody that is dissociated(if any) is freed from the antigen matrix and passes by elution with theeluent into the eluate.

In one embodiment, the material for the insoluble support (hereinafter"resin") takes the form of spherical beads. In a preferred embodiment,the resin is a synthetic polymer capable of forming a gel in aqueousmedia (e.g. agarose).

The immunoaffinity purification of the present invention provides anumber of benefits. First, the immunoaffinity purification of thepresent invention provides for maximum attachment of the antigen (e.g.venom) to the resin, i.e. high attachment efficiency. Second, theimmunoaffinity purification of the present invention provides for therecovery of as much of the reactive antibody of the unpurified antibody(the preferred unpurified antibody is PEG-purified whole yolk lgY) aspossible, i.e. the quantity of antibody purified is optimized. Third,the immunoaffinity purification of the present invention allows for therecovery of the antibody in an active state, i.e. the quality ofreactivity is preserved. Fourth, the immunoaffinity purification of thepresent invention provides that the bound antibody be elutedquantitatively; there is no significant (less than two percent) retainedantibody to progressively decrease column capacity after successivecycles of use, i.e. the antigen matrix is recyclable. Fifth, and mostimportantly, the immunoaffinity purification of the present inventionallows for the retention in the purified antivenom of the spectrum ofreactivity of the unpurified antivenom.

Most previous studies of polyclonal antibody purification have involvedmammalian antibodies. No comparative information is available on theforces affecting the stability of chicken antibody-antigen complexes.Indeed, very little structural or chemical data has been compiled on theproperties of chicken immunoglobulins (e.g., amino acid sequence ofheavy and light chains, disulfide bond structures, three-dimensionalstructure, freeze-thaw and thermal stability, etc.).

The majority of immunoaffinity purification of antivenom that has beendone has only used individual toxins and not whole venom. No comparativeinformation is available on the forces affecting the stability of thewhole venom antigen matrix.

The immunoaffinity purification of the present invention involvesconsideration of the venom(s) used to raise the antivenom that is to beimmunoaffinity purified. In this regard, the present invention providesa method for evaluating the immunochemical similarity or dissimilarityof venoms that allows for i) means of designing cost-effectiveimmunization cocktails for new antivenom formulas, ii) means ofdesigning cost-effective antigen matrices for purifying new or existingantivenoms, iii) means of identifying the monovalent and polyvalentantibody subpopulations of an existing antivenom, and iv) means ofdetermining its spectrum of reactivity of antivenom for the furtherdesign of antivenoms for treatment. Additional considerations includethe nature of the resin, method of binding the venom(s) to the resin,method of applying the unpurified antivenoms, and method of recoveringpurified antivenom.

i. Venoms Used To Immunize

Every geographic area has its own unique collection of venomous species.The greater the immunochemical similarity of the venoms produced bythese species, the greater the likelihood that antivenoms raised againstone species will react with and neutralize other species. The moredissimilar the venoms, the greater the need is for antivenoms that reactwith a number of species so that individual species need not beidentified in the case of an emergency.

Immunochemical similarity is better understood in terms of epitopes. Anepitope is defined as an antibody combining site on an antigen. Wherevenom is the antigen, an epitope is a discrete (typically measured inangstroms) region of a venom component where antibody binds to the venomcomponent. Where an epitope is not present in the venom of otherspecies, it is referred to as a "species-unique epitopes." Where anepitope is common to venom of different species it is referred to as a"species-shared epitope." Where there is a single species-unique epitopein a venom, there is said to be immunochemical dissimilarity between thevenom and any other venom. The greater the number of species-uniqueepitopes the greater the immunochemical dissimilarity.

Because of the practicality of treatment within one geographic area, thepresent invention contemplates raising antivenoms with a mixture ofvenoms (a "cocktail") as an immunogen. Using cocktails, the antivenomsof the present invention have reactivity with more than one venom.Furthermore, using the immunoaffinity purification of the presentinvention, the immunoaffinity purified antivenoms of the presentinvention retain this spectrum of reactivity. This is in contrast toexisting antivenoms. Previously, the purification of antivenoms raisedusing mixtures of venoms has not preserved the spectrum of reactivity ofthe unpurified antivenom.

The present invention contemplates that antivenom is a "population" ofantibodies. The population may be composed of one or more"subpopulations." Where more than one whole venom is used as animmunogen (and assuming the venoms are immunogenic), the resultingantivenom is "polyvalent." A polyvalent antivenom is herein defined as apopulation of antibodies having reactivity with all of the immunizingvenoms. Where only one whole venom is used as an immunogen, theresulting antivenom is "monovalent."

A polyvalent antivenom may have "monovalent subpopulations," "polyvalentsubpopulations," and/or "crossreactive subpopulations." In all cases,valency is defined by venom reactivity with immunizing venom(s) (and notby antibody class or subclass). Monovalent subpopulations of apolyvalent antivenom are herein characterized as subpopulationsexhibiting reactivity with some but not all immunizing venoms.Polyvalent subpopulations of a polyvalent antivenom are hereincharacterized as subpopulations exhibiting reactivity with all of theimmunizing venoms. Crossreactive subpopulations of a polyvalentantivenom are herein characterized as subpopulations exhibitingreactivity with non-immunizing venom(s).

Monovalent antivenoms may have "monovalent subpopulations" and"crossreactive subpopulations." Monovalent subpopulations of amonovalent antivenom are herein characterized as subpopulationsexhibiting reactivity with the immunizing venom. Crossreactivesubpopulations of a monovalent antivenom are herein characterized assubpopulations exhibiting reactivity with non-immunizing venom(s).

Subpopulation characterization is best understood by example and byignoring reactivity with non-immunizing venoms. Where a first and asecond whole venom are used together to immunize, the resultingpolyvalent antivenom can in theory have i) a monovalent subpopulation ofantibody reactive only with the first venom, ii) a monovalentsubpopulation of antibody reactive only with the second venom, and iii)a polyvalent subpopulation of antibody (i.e. antibody reactive with bothvenoms). On the other hand, the resulting polyvalent antivenom couldalso comprise two monovalent subpopulations without any polyvalentsubpopulation, or a polyvalent subpopulation without any monovalentsubpopulations.

Whether in fact the resulting polyvalent antivenom does have monovalentsubpopulations depends on whether the venoms used as immunogen havespecies-unique epitopes. Where there are no species-unique epitopes,there will be no monovalent subpopulations.

Polyvalent antivenom can be made either by a) immunizing with a venomcocktail or b) immunizing with single venoms and mixing two or moremonovalent antivenoms. In either case, the reactivity of thesubpopulation(s) determine the spectrum of reactivity of the population,i.e. the antivenom. Importantly, whether monovalent or polyvalent, thepurification of the present invention allows for the quantitativeretention in the purified antivenom of the spectrum of reactivity of theunpurified antivenom.

In nearly all previous studies, antitoxin and antivenom antibodies werepurified using only a single toxin. Antibodies purified in this mannerdo not have the spectrum of reactivity required to neutralize theplurality of distinct toxins present in whole venoms. In the handful ofstudies using whole venom, the investigators used only single venomantigen matrices. Single venom antigen matrices are not capable ofbinding and purifying the spectrum of antivenom antibodies present inthe polyvalent commercial antivenom investigated. Thus, purification inthe manner described by these researchers necessarily resulted inantivenom with a more limited reactivity than the unpurified antivenom.

To retain the spectrum of reactivity, the present invention givesconsideration to the venom(s) used to immunize when determining theappropriate venoms for immunoaffinity purification. In a preferredembodiment, the immunoaffinity purification of the present inventioncontemplates using the same venom or cocktail of venoms for purificationas was used for immunization. By using the same venom or cocktail ofvenoms, the purified antivenom derived from unpurified antivenom hassubstantially the same spectrum of reactivity as the unpurifiedantivenom, where "substantially" is defined as greater than 50% of theantigen-binding reactivity of the unpurified antivenom with respect toeach immunizing venom. As a relative value, the measurement of "50%reactivity" need not be made by any particular assay. Noetheless,conventional direct antigen-binding ELISA techniques are preferred. Inother embodiments, the present invention contemplates using differentvenom or cocktails for purification as was used for immunization.

ii. Nature of the Resin

The present invention contemplates immobilization of venoms with aninsoluble support. There are essentially three ways this can beachieved. First, venom components can be physically trapped in a gel;this approach does not rely upon any particular chemical reactivities ofthe venom components. Second, venom components can be covalently coupledto an "activated" matrix; this approach relies on the existence offunctional groups on the venom components that can covalently bond withthe matrix. Third, venom components can be coupled to insoluble supportsusing bifunctional reagents as linking groups that react with a) sidegroups on venom components and b) groups on the insoluble support.

As noted earlier, only individual venom components have thus far beencovalently attached to a matrix; whole venom has not been immobilized inthis manner. With single venom components, a number of factors,including mode of attachment, the type of resin, and the distancebetween the resin and the attached antigen, can influence the success ofthe immobilization approach. See e.g., V. Kukongviriyapan et al., J.Immunol. Meth. 49:97 (1982). With whole venoms these factors aremultiplied and result in greater uncertainty.

One important issue in immobilizing whole venom is coupling efficiency:are the functional groups accessible such that the bulk of the venomcomponents are coupled? Another issue involves antigenicity: doesattachment to the matrix preserve (or destroy) the antigenicity of thevenom components? With respect to the first issue, little is known aboutthe chemical structure of most venom components. While the existence ofreactive primary amines should allow for attachment to most activatedmatrices, there is no assurance that this will occur at a sufficientlevel to be useful. With regard to the second issue, it is possible thatthe functional groups involved in covalent attachment are part of, ornear, important epitopes. Random alteration or steric hindrance ofepitopes through covalent bonding of functional groups coulddramatically influence the antibody binding capacity of the matrix.Non-random alteration or steric hindrance of epitopes could be expectedto significantly impact the ability to recover immunoaffinity purifiedantivenom with the spectrum of reactivity of the unpurified antivenom.

In a preferred embodiment, the present invention provides a covalentattachment method for whole venom that allows for high couplingefficiency and high antibody binding capacity. It is preferred becauseof the ease and efficiency of antigen attachment, the stability of theattachment (relative to resins involving non-covalent attachment), andmechanical and chemical strength towards denaturants that are usedduring chromatographic procedures.

In one embodiment, the covalent attachment method employs cyanogenbromide activated Sepharose 4B (Pharmacia) as a resin for covalentattachment of whole venom. The preferred resins with active groups forcovalent attachment are resins with aldehydes as active groups("aldehyde-activated resins"), such as are described in U.S. Pat. No.3,836,433 to Wirth et al., which is hereby incorporated by reference. Apreferred resin is aldehyde-activated agarose. Chicken antibody elutedfrom such an antigen matrix exhibits a consistently higher venom bindingactivity (i.e. quality) than chicken antibody eluted from other antigenmatrices.

iii. Binding Venom(s) To The Resin

It is not intended that the immunoaffinity purification of the presentinvention be limited to any particular venom or mixture of venoms. Inthe preferred embodiment, however, a cocktail is used. The preferredbinding of the venom, whether single whole venoms or cocktails of wholevenom, to an aldehyde-activated resin is via sodium cyanoborohydridereduction.

In a preferred embodiment, the antigen matrix is "pre-stripped" with aneluent prior to any further use of the antigen matrix. By pre-stripping,the purification of the present invention avoids contamination ofimmunoaffinity purified antivenom preparations with venom that failed toattach to the resin.

iv. Applying Unpurified Antivenom

It was noted above that by using the same venom or cocktail of venoms inthe antigen matrix as was used in immunization, the purified antivenomderived from unpurified antivenom has substantially the same spectrum ofreactivity of the unpurified antivenom. The maximum retention of thespectrum of reactivity is achieved when one, in addition using the samevenom or cocktail of venoms in the antigen matrix as was used inimmunization, uses the venom or cocktail of venoms in the antigen matrixat a concentration that allows for the presentation of antigen ingreater amounts than that needed to bind all of the specific antibodyapplied to the antigen matrix. The latter is achieved by monitoring theflow through for reactivity as the unpurified antibody is applied; wherethe flow through shows less than 10% of the reactivity of the unpurifiedantibody that is applied, the venom or cocktail of venoms in the antigenmatrix is viewed to be at a concentration that allows for thepresentation of antigen in greater amounts than that needed to bind allof the specific antibody applied to the antigen matrix.

v. Recovering Purified Antivenom

In the preferred embodiment, the purification of the present inventioncontemplates sequential elution steps. By using sequential elutionsteps, the purification of the present invention overcomes the problemof poor recyclability that is associated with traditional purificationmethods.

In the preferred embodiment, the purification of the present inventionallows for quantitative purification of the venom-specific antibodiespresent initially in the egg yolk IgY, retention of antibodyantigen-binding activity, and recyclability of the matrix, by a firstelution with a non-denaturing eluent and a second elution withdenaturing eluent. The second elution recovers the remaining portion ofantibody and recycles the antigen matrix. Thus, following application ofcrude antivenom, a preferred embodiment of the method of the presentinvention comprises the steps: 1) washing the matrix with a firstbuffer, 2) washing the matrix with a second buffer, 3) washing thematrix with a third buffer, 4) dissociating specific antibody from thevenom with a first eluent, 5) washing the matrix again with the thirdbuffer, 6) stripping the column of substantially all non-venom proteinwith a second eluent, 7) re-equilabrating the column for recyclabilitywith the first buffer, and 8) removing eluent from the dissociated,specific antibody, wherein the first buffer is a phosphate-containing,low-ionic strength, non-detergent-containing, neutral pH buffer, thesecond buffer is a high ionic strength, non-ionic detergent-containing,slightly alkaline buffer, the third buffer is anon-phosphate-containing, low ionic strength, non-detergent-containing,neutral pH buffer, the preferred first eluent is a non-denaturingsolution compatable with an extended residence time to dissociate boundantibody, the preferred second eluent is a strongly denaturing solutionthat rapidly dissociates the antibody that was not released by the firsteluent, and the removal of both eluents from the dissociated antibody isperformed by extensive dialysis against a low ionic strength,non-detergent-containing, neutral pH buffer.

While not limited to any particular theory, it is believed that thefirst buffer washes the bulk of the unbound protein from the matrix. Thesecond buffer is of high ionic strength in order to washnon-venom-reactive, electrostatically-bound proteins from the matrix,and contains a non-ionic detergent to wash non-venom-reactive,hydrophobically-bound proteins from the matrix. Neither the high ionicstrength nor the non-ionic detergent should disrupt specificantibody-antigen interactions. The third buffer does not containphosphate because phosphate is not compatible with the preferred firsteluent. After the first eluent, it is highly desirable that the matrixbe equilibrated with the third buffer to wash the remaining eluent fromthe matrix before the second eluent is applied. After the second eluent,the phosphate-containing first buffer is used to remove the secondeluent and to re-equilibrate the matrix in preparation for a newpurification cycle.

In a preferred embodiment, the present invention contemplates 4Mguanidine-HCl pH 8.0 as a second eluent to dissociate theantigen-antibody complexes formed between the immobilized venom and thechicken antibody specific for the antigen, because of its efficiency,solubility at 4 C and ease of removal by dialysis.

It was determined that 0.1M glycine pH 2.5 as a first eluent causedunsatisfactory qualitative effects (high background sticking to solidsurfaces and aggregation of renatured antibody) on chicken antibodies.Similarly, 4M guanidine-HCl, when used as a first eluent, elutedmaterial that possessed only 17-49% of the original activity of theunpurified chicken IgY.

vi. Assessment of the Spectrum of Reactivity

As noted above, whether in fact the resulting polyvalent antivenom doeshave monovalent subpopulations depends on whether the venoms used asimmunogen have species-unique epitopes. The present inventioncontemplates determining whether a species' venom has species-uniqueand/or species-shared epitopes (relative to other species' venom). Thepresent invention contemplates making this determination using both i)antibody raised against single venoms (i.e. monovalent antivenoms), andii) antibody raised against a plurality of venoms (i.e. polyvalentantivenoms). In this manner, the antivenoms of the present invention areuseful in the selection of the appropriate venom and cocktails asimmunogens for the production of the preferred polyvalent antibodies fortreatment.

FIG. 2 shows schematically one approach to determining whether aspecies' venom has species-unique and/or species-shared epitopes. Inthis example, two antivenoms are used: one raised by immunization withVenom A and the other raised by immunization with Venom B. Depending onthe source of the antivenoms, a prestep (not shown) may be desirable toeliminate viscosity and hydrophobicity problems during purification(e.g. chicken antivenom derived from eggs should be PEG-treated in aprestep procedure to eliminate lipids). For ease of understanding, FIG.2 has been drawn to show species-unique epitopes (dark squares andcircles for Venom A and open squares and circles for Venom B) as well asspecies-shared epitopes (dark triangles).

Antivenom A and Antivenom B are immunoaffinity purified separately overtheir respective immunizing venom immobilized to make an antigen matrix(Matrix A and Matrix B). The flow-through in each case is reapplied toassure quantitative recovery and then discarded (dotted lines). Afterwashing, the bound antibody is eluted. The existence of antibody (ifpresent) is detected in each eluate (Eluate 1 is from Matrix A; Eluate 2is from Matrix B) by ultraviolet light absorption at 280 nm ("A₂₈₀ ")

The eluates in each case are then applied to the respectivenon-immunizing venom as an antigen matrix; Eluate 1 is applied to MatrixB' and Eluate 2 is applied to Matrix A' (Matrix A can be the same asMatrix A' or can be a duplicate; Matrix B can be the same as Matrix B'or can be a duplicate). Again, the flow-through in each case isreapplied to assure quantitative recovery (dotted lines). The finalflow-throughs are collected for characterization The existence ofnon-binding antibody in the flow-through is determined by A₂₈₀. Afterwashing, the bound antibody (if any) is eluted and collected forcharacterization. The existence of antibody (if present) is detected ineach eluate (Eluate 3 is from Matrix B'; Eluate 4 is from Matrix A') byA₂₈₀.

Where antibody is detected in the final flow-throughs, this antibodymust be non-crossreactive. This indicates there are one or morespecies-unique epitopes and consequently one or more monovalentsubpopulations. Where antibody is detected in the final eluates, thisantibody must be crossreactive. This indicates there are one or morespecies-shared epitopes and consequently one or more crossreactivesubpopulations.

FIG. 3 shows schematically a preferred approach to determining whether aspecies' venom has species-unique and/or species-shared epitopes. Inthis example, one antivenom is used; the antivenom was raised byimmunization with Venom A together with Venom B. Again, for ease ofunderstanding, FIG. 3 has been drawn to show species-unique epitopes(dark squares and circles for Venom A and open squares and circles forVenom B) as well as species-shared epitopes (dark triangles).

Antivenom AB is immunoaffinity purified sequentially over the immunizingvenoms used individually as antigen matrices (Matrix A, Matrix B andMatrix B'). The flow-throughs in all cases are reapplied (dotted lines)to assure quantitative recovery of specific antibody. The flow-throughfrom Matrix A (Flow-Through 1) is then applied to Matrix B. Afterwashing both Matrix A and Matrix B, the bound antibody is eluted. Theexistence of antibody (if present) in each eluate (Eluate I is fromMatrix A; Eluate 2 is from Matrix B) is detected by A₂₈₀.

Eluate 1 is applied to Matrix B' (Matrix B can be the same as Matrix B'or can be a duplicate). The flow-through (Flow-Through 2) is collectedfor characterization. The existence of non-binding antibody (if present)in the flow-through is determined by A₂₈₀. After washing Matrix B', thebound antibody (if any) is eluted and collected for characterization.The existence of antibody (if present) in this eluate (Eluate 3) isdetected by A₂₈₀.

Where antibody is detected in Eluate 2, this antibody must benon-reactive with Venom A, but reactive with Venom B. This indicatesthere are one or more species-unique epitopes and consequently one ormore monovalent subpopulations. Where antibody is detected in Eluate 3,this antibody must be reactive with both Venom A and Venom B. Thisindicates there are one or more species-shared epitopes and consequentlyone or more polyvalent subpopulations.

A comparison of FIG. 2 with FIG. 3 shows that the use of a cocktailimmunogen (FIG. 3) allows for the same determinations with fewer "runs"(i.e. elutions from antigen matrices). This advantage continues and,indeed, becomes more significant when epitope determinations are desiredfor greater numbers of venoms. For example, where three venoms need tobe analyzed, a cocktail immunogen approach allows for epitopedeterminations with only seven runs, while a single venom immunogenapproach requires twelve runs.

Once epitope determinations are made for the venoms of interest, apreferred cocktail matrix can be designed for immunoaffinitypurification such that the purified antivenom retains the spectrum ofreactivity of the unpurified antivenom. While a cocktail matrixcontaining all of the immunizing venoms will (if used in a quantitativeprotocol) invariable retain the spectrum of reactivity (see FIG. 4),elimination of venoms having no species-unique epitopes may be desiredwhere large-scale (e.g. commercial) purifications are to be performedwith scarce and/or expensive venom(s).

III. Treatment

The present invention contemplates antivenom therapy for envenomedhumans and animals. The method of antivenom treatment of the presentinvention involves consideration of a) venom identification, b) degreeof envenomation, and c) type and dose of antivenom to be administered.

A. Venom Identification

Commonly, the venomous species is not seen, let alone captured foridentification, at the time of envenomation. The lack of reliablespecies identification, particularly in emergency situations, takentogether with the cost of raising antivenom, makes it preferrable thatthe antivenoms used in treatment not be limited in their reactivity to asingle species. Thus, in a preferred embodiment, the present inventioncontemplates raising polyvalent antivenoms according to the potentialfor envenomation by venomous inhabitants of any particular geographicalarea.

B. Degree of Envenomation

Not all venoms are potentially fatal; even bites or stings from the mostpotent species may not be life-threatening if a relatively low degree ofenvenomation occurs. A key clinical dilemma, however, results from thefact that the amount of venom delivered is highly variable and theattending medical personnel must rely on the victim's symptoms inassessing the extent of the overall threat of serious injury or death.These symptoms include, but are not limited to, local pain,hemorrhaging, numbness, edema, necrosis, nausea, vomiting, bloodclotting abnormalities, faintness, proteinuria, respiratory distress andparalysis. Importantly, the severity of these symptoms must beconsidered in connection with the time after envenomation. Typically,symptoms are more severe over time. Thus, less severe symptoms early ondo not ensure a low level of envenomation. The qualitative nature ofthese symptoms and the frequent difficulty in establishing a meaningfultime frame make a determination of the degree of envenomationapproximate at best.

C. Dosage of Antivenom

It was noted by way of background that a balance must be struck whenadministering currently available antivenom; sufficient antivenom mustbe administered to neutralize the venom, but not so much antivenom as toincrease the risk of untoward side effects. These side effects arecaused by i) patient sensitivity to horse proteins, ii) anaphylactic orimmunogenic properties of non-immunoglobulin proteins, iii) thecomplement fixing properties of mammalian antibodies, and/or iv) theoverall burden of foreign protein administered. It is extremelydifficult to strike this balance when, as noted above, the degree ofenvenomation (and hence the level of antivenom therapy needed) can onlybe approximated.

The present invention contemplates significantly reducing side effectsso that this balance is more easily achieved. Treatment according to thepresent invention contemplates reducing side effects by using i)immunoaffinity purified antivenom from mammalian sources, ii)PEG-purified antivenom from birds, and/or iii) immunoaffinity purifiedantivenom from birds

In one embodiment, the treatment of the present invention contemplatesthe use of immunoaffinity purified antivenom from mammalian sources.While complement-fixing, immunoaffinity purification of antivenom frommammalian sources reduces the total protein burden up to approximatelytwenty fold. This means that approximately twenty times morevenom-reactive antibody can be administered before the risk of serumsickness reaches that of the currently available antivenom.

In another embodiment, the treatment of the present inventioncontemplates the use of PEG-purified antivenom from birds. The use ofyolk-derived, PEG-purified antibody as antivenom allows for theadministration of 1) non(mammalian)--complement-fixing, avian antibody,2) a less heterogeneous mixture of non-immunoglobulin proteins, and 3)only one-third as much total protein to deliver the equivalent weight ofactive antibody present in currently available antivenom. This meansthat approximately three times more venom-reactive antibody can beadministered before the risk of serum sickness reaches that of thecurrently available antivenom. The non-mammalian source of the antivenommakes it useful for treating patients that are sensitive to horse orother mammalian serums. PEG-purified antivenom is useful for treatingvictims where the amount of antivenom required is relatively small orthe cost of affinity purification is prohibitive. The amount ofantivenom required may be relatively small (<250 mg) when the extent ofsystemic envenomation is slight. For instance, low envenomation mayoccur in the case of certain scorpion species, very small or immaturesnakes, or snake species that typically deliver small (<5 mg) of venomin a single bite. Cost is prohibitive in certain regions of the world;such areas cannot sustain the financial burden of immunoaffinityreagents and the skilled labor necessary to produce highly-purifiedantivenoms.

In a preferred embodiment, the treatment of the present invention usesyolk-derived, immunoaffinity purified antibody as antivenom, whichallows for the administration of 1) non-complement-fixing antibody, and2) only 1/20th as much total protein to deliver the equivalent weight ofactive antibody present in currently available antivenom. This meansthat twenty times more venom-reactive antibody can be administeredbefore the risk of serum sickness reaches that of the currentlyavailable antivenom. Thus, physicians may treat victims moreaggressively with far greater amounts of active antivenom without fearof increased side effects. Because the initial foreign protein exposureis reduced, the risk of sensitizing a patient to antivenom is alsoreduced. This is important where the patient must undergo subsequentantivenom treatment.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: A₂₈₀(Absorbance at 280 nm); eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles);; nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); °C. (degrees Centigrade); CFA(Complete Freund's Adjuvant); IFA (Incomplete Freund's Adjuvant); ELISA(Enzyme-linked Immunosorbent Assay); MW (molecular weight); 0D (opticaldensity); EDTA ethylene-diaminetetracetic acid); PAGE (polyacrylamidegel electrophoresis); Aldrich (Aldrich Chemical Co., Milwaukee, Wis.);Beckman (Beckman Instruments, San Ramon, Calif.); BRL (Bethesda ResearchLaboratories, Gaithersburg, Md.); Cappel (Cappel Laboratories, Malvern,Pa.); Eastman (Eastman Kodak, Rochester, N.Y.); Fisher (Fisher Biotech,Springfield, N.J.); GIBCO (GIBCO, Grand Island, N.Y.); Gilford (Gilford,Oberlin, Ohio); IBF (IBF Biotechnics, Savage, Md.); Mallinckrodt(Mallinckrodt, St. Louis, Mo.); Pierce (Pierce Chemical Co., Rockford,Ill.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Wyeth (WyethLaboratories, Marietta, Pa.).

For convenience when discussing antivenoms, the immunized animal used asthe source is used as a modifier in front of the term "antivenom" (e.g."horse antivenom" means antivenom raised in a horse and "chickenantivenom" means antivenom raised in a chicken).

The antivenom used as starting material and/or control antivenom in someexamples below (hereinafter "unpurified horse antivenom") was obtainedfrom Wyeth (lot #M878035) This unpurified horse antivenom has been usedextensively by others for the treatment of humans afflicted withvenomous bites. D. C. Christopher and C. B. Rodning, S. Med. J. 79:159(1986). M. J. Ellenhorn and D. G. Barceloux, Medical Toxicology, Ch.39(Elsevier Press 1988). H. M. Parrish and R. H. Hayes, Clin. Tox. 3:501(1970). F. E. Russell et al., JAMA 233:341 (1975).

EXAMPLE 1 Production of an Antivenom to a Cocktail of Modified SnakeVenoms in a Non-Mammal

To determine the best course for raising high titer egg antibodiesagainst venoms, the effect of various methods of venom modification wasexamined. In order to demonstrate that, as a result of modification,toxicity would be inactivated but antigenicity would be preserved. Theexample involved a) venom modification, b) immunization, c) antivenomcollection, and d) antigenicity assessment.

a) Venom modification: Crotalus atrox venom (Sigma) was modified byformaldehyde, glutaraldehyde or heat treatment. In order to monitor theinactivation of venom, the inhibition of total venom protease activitywas measured according to a modified method of V. B. Philpot, Jr. etal., Toxicon 16, 603 (1978). The assay consisted of mixing 10microliters of venom or buffer control with 25 microliters ofazo-coupled cowhide beads (Sigma) in 200 microliters of PBS for five tofifteen minutes. Protease activity is detected when the supernatantturns red due to hydrolysis of the azo dye. Results were quantitated atA₅₂₅ on a spectrophotometer (Gilford).

With respect to inactivation, it is not necessary that completeinhibition be achieved. It is simply desirable as a means of minimizingthe impact of immunization in the immunized animal.

For formaldehyde treatment, a 10 mg/ml dilution of whole C. Atrox venomwas made in various concentrations of formaldehyde (0.25-8.0% w/v) andleft for 1 hour at room temperature. Because it was observed that 8.0%formaldehyde gave complete inhibition of venom protease activity, theseconditions were used to prepare the formaldehyde-treated immunogen.

For glutaraldehyde treatment, a 10 mg/ml dilution of whole C. Atroxvenom was made in glutaraldehyde and left for 1 hour at roomtemperature. Because it was observed that 1% glutaraldehyde totallyinactivated venom protease activity, this concentration was used toprepare the formaldehyde-treated immunogen.

For heat treatment, a 10 mg/ml dilution of whole C. Atrox was heated to95° C. in a water bath for five minutes and then plunged into ice. Thistreatment abolished all protease activity and was, therefore, thetreatment used to prepare heat-treated immunogen.

b) immunization: Two, one-year old white leghorn hens were eachimmunized with 1 mg of Crotalus atrox venom after it was treatedaccording to one of the inactivation methods described above (total ofsix immunized hens). The hens were injected with the inactivated venomin CFA (GIBCO) on day zero subcutaneously in multiple sites (both sidesof the abdomen, both breasts, and in both wings to involve more of thelymphatic system). The hens were subsequently injected with 1 mg of thesame inactivated venom in IFA (GIBCO) on Day 11 and Day 19.

c) antivenom collection: Chicken immunoglobulin (IgY) was extractedaccording to a modification of the methods of A. Polson et al., Immunol.Comm. 9:495 (1980). A gentle stream of distilled water from a squirtbottle was used to separate the yolks from the whites, and the yolkswere broken by dropping them through a funnel into a graduated cylinder.The broken yolks were blended with 7 volumes of egg extraction buffer toimprove antibody yield (Egg extraction buffer =0.01M Sodium phosphate,0.1M NaCl, pH 7.5, containing 0.01% NaN₃), and PEG 8000 (Baker) wasadded to a concentration of 3.5%. When all the PEG dissolved, theprotein precipitate that formed was pelleted by centrifugation at 9000×gfor 10 minutes. The supernatant was decanted and filtered throughcheesecloth to remove the lipid layer, and PEG was added to a finalconcentration of 12% (we assumed the supernatant was 3.5% PEG). Thesolution was centrifuged as above, the supernatant discarded, the pelletredissolved in 2 times the original yolk volume of egg extractionbuffer, and PEG added to 12% for a second precipitation. Aftercentrifugation the supernatant was discarded and the pellet centrifugedtwice more to extrude the PEG. This crude IgY pellet was then dissolvedin the original yolk volume of egg extraction buffer and stored at 4° C.

d) antigenicity assessment: Eggs were collected during the day 25--day28 period (and stored intact at 4° C. for approximately six months) toassess whether the venoms were sufficiently antigenic to raise anantibody. Eggs from the two hens in each modification group were pooledand antibody was collected as described above. Antigenicity was assessedon Western Blots according to the method of H. Towbin et al. Proc. Nat.Acad. Sci. USA 76:4350 (1979). 100 μg samples of three distinct venomtypes (Crotalus adamanteus, Crotalus atrox, and Agkistrodon piscivorus)were dissolved in SDS reducing sample buffer (1% SDS, 0.5%2-mercaptoethanol, 50 mM Tris pH 6.8, 10% glycerol, 0.025% w/vbromophenol blue), heated at 95° C. for 10 minutes and separated on a 1mm thick, 12% SDS-polyacrylamide gel. K. Weber and M. Osborn, In: TheProteins, 3rd Edition (H. Neurath and R. L. Hill, Eds.) (Academic Press,NY) (pp. 179-223). Part of the gel was cut off and the proteins stainedin Coomassie Blue. The proteins in the remainder of the gel weretransferred to nitrocellulose using the ABN polyblot electro-blottingsystem according to the manufacturers instructions (Fisher). Thenitrocellulose was temporarily stained with 10% Ponceau S (S. B. Carrolland A. Laughon, In: DNA Cloning: A Practical Approach, Vol. III, D.Glover, Ed., IRL Press, Oxford, pp.89-111)) to visualize the lanes, thendestained by running a gentle stream of distilled water over the blotfor several minutes. The nitrocellulose was immersed in PBS containing3% BSA overnight at 4.C to block any remaining protein binding sites.

The blot was cut into strips and each strip was incubated with theappropriate primary antibody. The three primary antibodies discussedabove were used (along with pre-immune chicken antibody as a control)diluted 1:250 in PBS containing 1 mg/ml BSA for 2h at room temperature.The blots were washed with 2 changes each of large volumes of PBS,BBS-Tween and PBS successively (10 min/wash). Goat anti-chicken IgGalkaline phosphatase conjugated secondary antibody (Fisher Biotech) wasdiluted 1:400 in PBS containing 1 mg/ml BSA and incubated with the blotfor 2 hours at room temperature. The blots were washed with 2 changeseach of large volumes of PBS and BBS-Tween, followed by 1 change of PBSand 0.1M Tris-HCl, pH 9.5. Blots were developed in freshly preparedalkaline phosphatase substrate buffer: 100 μg/ml Nitro-Blue Tetrazolium(Sigma), 50 μg/ml 5-Bromo-4-Chloro-3-Indolyl Phosphate (Sigma), and 5 mMMgCl₂ in 50 mM Na₂ CO₃ pH 9.5.

The results are shown in FIG. 5. The Coomassie Blue strip (Strip 1)illustrates the order of the venoms put in all the other strips:Crotalus adamanteus was placed in Lane 1; Crotalus atrox, was placed inLane 2; Agkistrodon piscivorus was placed in Lane 3. From FIG. 5,antibody reactivity is seen in Strips 2 (formaldehyde-treated immunogen)and 4 (heat-treated immunogen); very little reactivity can be seen inStrips 3 (glutaraldehyde-treated immunogen) and 5 (no immunogen). Thissuggests that, while the glutaraldehyde treatment was useful to reduceprotease activity, the treatment denatured the venom to the point whereantigenicity was also severely reduced. In terms of antigenicity, itappears from FIG. 5 that there is the following relationship among thedifferent treatments:heat-treated>formaldehyde-treated>glutaraldehyde-treated.

Importantly, the chicken antibody recovered from the eggs reacts withmany protein bands in Lane 2 (the C. atrox venom preparation) of Strips2 and 4. The presence of many protein bands illustrates the complexityof the various venoms. Some of these protein bands from the differentvenoms appear to co-migrate, suggesting that there might be proteins incommon among the venoms. Interestingly, while the antivenom was raisedagainst a single venom (C. atrox), the results from Lanes 1 (theCrotalus adamanteus preparation) and 3 (the Agkistrodon piscivoruspreparation) of Strips 2 and 4 indicate that the chicken antibody reactswith non-immunizing venoms, i.e. is crossreactive. Clearly, there is aset of antigenically related proteins in the three different venoms.

EXAMPLE 2 Adjuvant Effects on Antivenom Titers

To determine the best course for raising high titer egg antibodiesagainst venoms, the impact of different types of adjuvants wasdemonstrated. The example consisted of a) adjuvant/antigen mixturepreparation, b) immunization, c) antibody collection, and d)antigenicity assessment.

a) adjuvant/antigen preparation: In all cases the antigen consisted of 1mg of each of 4 snake venoms (4 mg of total antigen per bird):Agkistrodon contortrix, Agkistrodon piscivorus, Crotalus atrox, andCrotalus adamanteus (Sigma).

To prepare the Ribi adjuvant/venom antigen mixture, three volumes of theheat-inactivated antigen (heat inactivation was as described inExample 1) in PBS were mixed with one volume of Ribi LES+STM adjuvant(Ribi ImmunoChem Research Inc., Hamilton, Mont.) at 37° C. and vortexedto a milky emulsion before injection.

To prepare the Freund's adjuvant/venom antigen mixture, heat-inactivatedvenom was mixed in with CFA (GIBCO) in a relationship of 5:4(adjuvant:antigen by volume) and emulsified to a firm consistency bypassage through an antigen mixer made from two 18 gauge stainless steelhypodermic needles that had been brazed together.

To prepare the bentonite adjuvant/venom antigen mixture, one volume ofnative venom was mized with one volume of a sterile, 2% (w/v) bentonite(Sigma) suspension to adsorb the venom proteins to the particulate.

b) immunization: Six, (previously unimmunized) one-year old whiteleghorn hens (numbered for reference as #337, #339, #340, #353, #354,and #355) were immunized on Day zero. Two birds (#339, #354) receivedthe Ribi adjuvant/antigen mixture. Two other birds (#337, #353) receivedthe antigen with CFA. The remaining two birds (#340, #355) received theantigen absorbed to bentonite. The hens were injected subcutaneously inmultiple sites (both sides of the abdomen, both breasts, and in bothwings to involve more of the lymphatic system).

All of the birds were re-injected in the same manner with the sameamount of antigen (prepared in the same way for each two bird group) onDays 14 and 21, with the exception of birds #337 and #353, whichreceived antigen in IFA.

c) antibody collection: Antibody was extracted from the eggs asdescribed in Example 1. Importantly, the method of extracting antibodyfrom the eggs resulted in approximately 80% recovery of initial antibodyrecovery according to the following assay:

Yolks were blended with seven volumes of egg extraction buffer (0.01M Naphophate, 0.1M NaCl, pH 7.5 containing 0.01% NaN₃). Then a small volumeof diluted yolk was further diluted seven-fold in egg extraction buffer,centrifuged at 9000×g for 10 minutes and the supernatant assayed forantibody activity by ELISA [see d) below]. PEG-purified material wascompared with the crude yolk sample for antivenom activity.

Eggs were collected from bird #355 beyond Day 28 for later use;PEG-purified antibody from #355 eggs collected from days 31-45 isreferred to as "PEG-purified 355" in Examples 13, 16, 25, and 27, below.

d) antigenicity assessment: The impact of the different adjuvants on theantigenicity of the venom was assessed on Day 28 eggs (stored intact at4° C. until they were assayed on Day 40) by ELISA. To prepare for theELISA, 96-well Nunc Immuno-Plates were coated overnight at 4° C. in ahumidified chamber with 200 μl/well of the appropriate venom (in thiscase C. atrox) at a concentration of 2 μg/ml. The next day the wellswere blocked with PBS containing 0.1% bovine serum albumin (BSA) for 2hours at room temperature. To perform the ELISA, appropriately dilutedantibody was added in PBS containing 0.1% BSA and the plates wereincubated for 2 hours at room temperature. The plates were then washedthree times with BBS (0.1M boric acid, 0.025M sodium borate, 1M NaCl, pH8.3) containing 0.1% Tween 20, twice with PBS containing 0.1% Tween, andtwice with just PBS. Alkaline phosphatase-conjugated rabbit anti-chickIgG (Fisher) was diluted 1:500 in PBS containing 0.1% BSA, added to theplates, and incubated 2 hours at room temperature. The plates werewashed as before, except Tris-buffered saline, pH 7.2, was substitutedfor PBS in the last wash, and p-nitrophenyl phosphate (Sigma) was addedat 1 mg/ml in 0.05M Na₂ CO₃ pH 9.5, 10 mM MgCl₂. The plates were thenevaluated either qualitatively by visual examination or quantitativelyby reading at 410 nm on a Dynatech MR300Micro ELISA reader approximately30 minutes after the substrate was added.

The ELISA results from the Day 28 eggs showed good reactivity for allthe birds (data not shown). However, no clear difference between thethree adjuvants was apparent when evaluated qualitatively. Nonetheless,bentonite did cause a palpable abcess in one bird, consistent with itsreported tendency to do so. P. A. Christensen, In: Snake Venoms(Springer-Verlag 1979), Chapter 20 (pp. 825-846). Bentonite also causeda decrease in the laying frequency of this bird.

In view of the cost of the RIBI mixture and the side-effects of thebentonite, the fact that Freund's adjuvant works just as well makesFreund's adjuvant a preferred adjuvant.

EXAMPLE 3 Booster Immunizations with a Cocktail of Native Venoms

To optimize the response and increase the titer of antibody, furtherimmunization was demonstrated. The example involved a) adjuvant/antigenmixture, b) immunization, c) antivenom collection, and d) antibody titerassessment.

a) adjuvant/antigen mixture: since this example involved the use ofimmunized birds (contrast Examples 1 and 2), the venoms were notmodified and were used in their native form. The venom mixture consistedof 0.5 mg each of Crotalus atrox and Crotalus adamanteus and 0.25 mg ofB. atrox (Sigma). IFA was mixed with the venom mixture in a 5:4 volumeratio (adjuvant:antigen) and emulsified to a firm consistency by passagethrough an antigen mixer made from two 18 guage stainless steelhypodermic needles that had been brazed together. b) immunization: Thesix one-year old white leghorn hens of Example 2 (#337, #339, ·340,#353, #354, and #355) were immunized on Day 49. All the birds receivedthe same adjuvant/antigen mixture. As before, the hens were injectedsubcutaneously in multiple sites.

c) antibody collection: Antibody was collected from the eggs asdescribed in Example 1.

d) antibody titer assessment: The impact of the Day 49 boost of nativevenom (including B. atrox for the first time) on antibody titer wasassessed on Day 62 using Day 57-61 eggs (stored intact at 4° C. untilthey were assayed on Day 62) by ELISA as described in Example 2.

The results are shown in FIG. 6. Chicken #354 clearly had the highesttiter as measured on Day 60 following the Day 49 boost. Interestingly,the bird with the lowest titer, #339, was previously immunized in thesame manner as #354, suggesting that other factors may be involved ingenerating high titers than immune status. Importantly, all the birdsshow a significant titer as compared with the unimmunized control.

EXAMPLE 4 Response of a Non-Mammal to High Doses of a Cocktail of NativeVenoms

To optimize the response and increase the titer of antibody, furtherimmunization was demonstrated. In this example, bird #354 was usedexclusively. As in Example 3, the example involved a) adjuvant/antigenmixture, b) immunization, c) antivenom collection, and d) antibody titerassessment.

a) adjuvant/antigen mixture: As in Example 3, this example involved theuse of immunized birds (contrast Examples 1 and 2). Therefore, thevenoms were not modified and were used in their native form. The firstvenom mixture consisted of 0.5 mg each of Crotalus atrox and Crotalusadamanteus and 0.5 mg of B. atrox (Sigma). The second venom mixtureconsisted of 2.5 mg each of Crotalus atrox and Crotalus adamanteus(Sigma). The third venom mixture consisted of 10 mg each of Crotalusatrox and Crotalus adamanteus (Sigma). The first, second and third venommixtures were mixed separately with IFA. The three adjuvant/antigenmixtures were mixed and emulsified as in Example 3.

b) immunization: Bird #354 was immunized on Day 72 with the firstadjuvant/antigen mixture, on Day 86 with the second adjuvant/antigenmixture, and on Day 106 with the third adjuvant/antigen mixture. In allcases, the injections were made subcutaneously in multiple sites.

c) antibody collection: Antibody was extracted from yolks (as describedin Example 1) of Day 58-60 eggs (hereinafter "PEG-purified Pool 1";PEG-purified Pool 1 is used in Example 18, below), Day 74-81 eggs(hereinafter "PEG-purified Pool 2"); PEG-purified Pool 2 is used inExamples 16, 17, 18 and 19 below), Day 94-99eggs (hereinafter"PEG-purified Pool 3"; PEG-purified Pool 3 is used in Examples 18 and 28below) and Day 120-126 eggs (hereinafter "PEG-purified Pool 4";PEG-purified Pool 4 is used in Examples 5, 14, 18, 23 and 26).

d) antibody titer assessment: The impact of the Day 72 boost of nativevenom (including an increased dose of B. atrox) on antibody titer wasassessed on Day 81 using Pool 2 (the eggs were stored intact at 4° C.until they were extracted and assayed on Day 81) by ELISA (see Example 2for general discription of ELISA). The results indicated a continuingincrease in antibody titer.

Importantly, the previously immunized birds tolerated 20 mg of activenative venom (3 times the dose required to kill an adult human on a bodyweight basis) with no apparent ill effects. This demonstrates that fargreater immunizing doses (mg/kg) can be used than have been used inimmunization schedules in the past. These higher doses allow for higherantivenom titers.

EXAMPLE 5 Duration of the High Titer Response to Venoms in a Non-Mammal

To optimize the duration of the response and increase the productiveperiod of the laying hen, further immunization was demonstrated. In thisexample, bird #354 was used exclusively. As in Example 4, the exampleinvolved a) adjuvant/antigen mixture, b) immunization, c) antivenomcollection, and d) antibody titer assessment.

a) adjuvant/antigen mixture: As in Example 4, this example involved theuse of immunized birds. Therefore, the venoms were not modified and wereused in their native form. The first venom mixture consisted of 2.5 mg.each of Crotalus atrox and Crotalus adamanteus (Sigma); the second venommixture consisted of 5.0 mg. each of C. atrox and C. adamanteus: thethird mixtures consisted of 10 mg. C. atrox and 5 mg. C. adamanteus: thefourth mixture consisted of 10 mg. C. atrox and 5 mg. C. adamanteus: thefifth mixture consisted of 15 mg. C. atrox and 10 mg. C. adamanteus. Allfive venom mixtures were mixed separately with IFA and theadjuvant/antigen mixtures emulsified as in Example 3.

b) immunization: Bird #354, which had been last immunized on day 106(see Example 4), was immunized on day 300 with the firstadjuvant/antigen mixture, on day 328 with the second adjuvant/antigenmixture, on day 356 with the third adjuvant/antigen mixture, on day 372with the fourth adjuvant mixture, and on day 407 with the fifthadjuvant/antigen mixture. In all cases, the injections were madesimultaneously in multiple sites.

c) antivenom collection: Antivenom antibody was extracted from the eggsas described in Example 1.

d) antibody titer assessment: The impact of these five boosts of nativevenom was assessed on day 422 using antibody from day 412-415 eggs ("day422 prep" indicates the eggs were stored intact at 4.C until antibodywas PEG-purified and assayed on day 422) and compared in an ELISA (seeExample 2) with PEG-purified Pool 4.

It can be seen (FIG. 7) that the response of the bird is comparable withboth preparations (in terms of reactive antivenom per ml). Thus, a bird,that has not been immunized for almost two hundred days, can bere-immunized with venom in a second immunization program such that theresponse is equivalent to the response observed after the initialimmunization program. Clearly, birds are useful for more than one yearfor antivenom production.

EXAMPLE 6 Covalent Attachment of Whole Snake Venom to CyanogenBromide-Activated Agarose Matrix

In this example, the coupling efficiency of Sepharose 4B (Pharmacia)(hereinafter "resin I"), was demonstrated using snake venom as antigen.C. atrox venom was diluted in PBS (pH 7.2) at a concentration of 10mg/ml. In a chemical hood, Resin I was washed with 5 volumes of chilleddistilled H₂ O, suspended in an equal volume of 2.5M potassium phosphatebuffer (pH 12.2) in a beaker immersed in an ice bath, and stirredgently. In a separate vessel in the same chemical hood, 1 gram of CNBr(Alrich) was dissolved in 1 ml of acetonitrile per 10 ml of gel to becoupled. Thereafter, the CNBr solution was added to the gently stirringsolution of resin I over a period of two minutes. The mixture(hereinafter "activated resin I") was continually stirred for anadditional eight minutes and then washed in a scintered glass funnelwith 10 volumes of cold distilled H₂ O followed by 10 volumes of coldPBS. The venom solution was then added to the activated resin I in thefunnel and the mixture (hereinafter "antigen matrix") was agitatedovernight. The uncoupled filtrate was collect from the funnel andmeasured (A₂₈₀) Coupling efficiency was calculated as the amount ofcoupled protein (A₂₈₀ units) divided by the total starting amount ofprotein (A₂₈₀). The results showed that the coupling efficiency ofactivated resin I was in the range of 90-95%. Thus almost 10 mg of venomprotein was bound per ml of resin.

For later use, the antigen matrix was suspended in an equal volume of 1Methanolamine-10mM Tris-HCl (pH 8.5) for 2 hours at 4° C. to blockremaining protein-reactive sites. The antigen matrix was then washedwith PBS containing 0.02% sodium azide and stored at 4° C.

EXAMPLE 7 Covalent Attachment of Whole Snake Venom to anAldehyde-Activated Polyacrylamide/Agarose Matrix

In this example, the coupling efficiency of the aldehyde-activated,polyacrylamide/agarose resin, Ultrogel AcA 22 (IBF) (hereinafter"activated resin II"), was demonstrated. C. atrox venom was diluted inPBS (pH 7.2) at a concentration of 10 mg/ml. Activated Resin II waswashed with 10 volumes of distilled H₂ O and then with 2.5 volumes of0.5M NaPO₄ (pH 7.0). Resin II was then added in an equal volume to thevenom solution. The mixture (hereinafter "antigen matrix") was splitinto two equal volumes. One was agitated for 18 hours at 4° C. The otherwas agitated for 18 hours at room temperature. Both antigen matrixsolutions were washed with PBS on a glass funnel. The filtrates werecollected and coupling efficiency was calculated as in Example 6. Theantigen matrix agitated at 4° C. showed 52% coupling yield and theantigen matrix agitated a room temperature showed 62% coupling yield.Thus, 5-6 mg of venom protein per ml of matrix was coupled usingactivated Resin II.

For later use, protein reactive sites were blocked in 1Methanolamine-10mM Tris-HCl (pH 8.0) at 4° C. for 3 hours. The antigenmatrix was then washed and stored in PBS containing 0.02%.

EXAMPLE 8 Covalent Attachment of Whole Snake Venom to anAldehyde-Activated Agarose Matrix

In this example, the coupling efficiency of the aldehyde-activatedresin, ACTIGEL A (Sterogene) (hereinafter "activated resin III"), wasdemonstrated. C. atrox venom was dissolved in PBS (pH 7.2) at aconcentration of 10 mg/ml. Activated Resin III was washed with 3 volumesof PBS and added (in equal volume) to the venom solution. Thereafter,1/10 volume of 1M sodium cyanoborohydride (Aldrich) was added. Themixture (hereinafter "antigen matrix") was then split into two equalvolumes. One was agitated for 4 hours at room temperature. The other wasagitated overnight at 4° C. Both antigen matrix mixtures were washed onglass funnels with PBS. The filtrate was collected and couplingefficiency was calculated as in Example 6. The results showed thatcoupling efficiency of activated Resin III is in the range of 80 to 90%.The antigen matrix was stored in PBS containing 0.02% sodium azide at 4°C.

EXAMPLE 9 Covalent Attachment of Whole Snake Venom from a Second Speciesto an Aldehyde-Activated Agarose Matrix

In this example, the coupling efficiency of the aldehyde-activatedresin, ACTIGEL A (Sterogene) (hereinafter "activated resin III"), wasstudied with another venom. C. durissus terrificus venom was dissolvedin PBS (pH 7.2) at a concentration of 5 mg/ml. Activated Resin III waswashed with 3 volumes of PBS and added (in equal volume) to the venomsolution. Thereafter, 1/10 volume of 1M sodium cyanoborohydride(Aldrich) was added. The mixture (hereinafter "antigen matrix") wasagitated overnight at 4° C. The antigen matrix was then washed on aglass funnel with PBS. The filtrate was collected and couplingefficiency was calculated as in Example 6. The results showed thatcoupling efficiency of activated Resin III was 95%. The antigen matrixwas stored in PBS containing 0.02% sodium azide at 4° C.

EXAMPLE 10 Covalent Attachment of Whole Snake Venom from a Third Speciesto an Aldehyde-Activated Agarose Matrix

In this example, the coupling efficiency of the aldehyde-activatedresin, ACTIGEL A (Sterogene) (hereinafter "activated resin III"), wasstudied with another venom. C. adamanteus venom was dissolved in PBS (pH7.2) at a concentration of 10 mg/ml. Activated Resin III was washed with3 volumes of PBS and added (in equal volume) to the venom solution.Thereafter, 1/10 volume of 1M sodium cyanoborohydride (Aldrich) wasadded. The mixture (hereinafter "antigen matrix") was agitated for 4hours at room temperature. The antigen matrix was then washed on a glassfunnel with PBS. The filtrate was collected and coupling efficiency wascalculated as in Example 6. The results showed that coupling efficiencyof activated Resin III was 78%. The antigen matrix was stored in PBScontaining 0.02% sodium azide at 4° C.

EXAMPLE 11 Covalent Attachment of a Mixture of Whole Snake Venoms to anAldehyde-Activated Agarose Matrix

In this example, the coupling efficiency of the aldehyde-activatedresin, ACTIGEL A (Sterogene) (hereinafter "activated resin III") wasstudied with a cocktail of four venoms. C. atrox, C. adamanteus, A.piscivorus and A. contortrix venoms were dissolved together in PBS (pH7.2) with each venom at a concentration of 10 mg/ml. Activated resin IIIwas washed with 6 volumes of PBS and added (in equal volume) to thevenom solution. Thereafter, 1/10 volume of 1M sodium cyanoborohydride(Aldrich) was added. The mixture (hereinafter "antigen matrix") was thenagitated for seven hours at room temperature and left overnight at 4° C.The antigen matrix was then washed on a glass funnel with PBS. Thefiltrate was collected and coupling efficiency was calculated as inExample 6. The results showed that coupling efficiency of activatedresin III was 54%. The antigen matrix was stored in PBS containing 0.02%sodium azide at 4° C.

EXAMPLE 12 Elution of Specifically-Bound Antibodies from an AffinityMatrix with Different Eluents

In this example, the elution efficiency of various eluents on chickenantibody is demonstrated. Chicken antibody is generated, collected andextracted from eggs as described in Example 1. Resin I is used toprepare an antigen matrix as in Example 6. Five eluents are studied insuccessive elutions. Between each elution, the antigen matrix is washedwith TBS, the eluate is collected and measured (A₂₈₀), the antigenmatrix is stripped of remaining antibody with 4M guanidine-HCl, thisstripped antibody is collected and measured (A₂₈₀), the antigen matrixis washed and the same amount of PEG-purified, chicken antibody (dilutedin egg extraction buffer) is loaded on the antigen matrix at a flow rateof 1 ml per min, and washed successively with several bed volumes ofPBS, BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1M NaCl, 0.1%(v/v) Tween 20 pH 8.3), and PBS until the effluent is free of protein(A₂₈₀)

In the first elution, bound chicken antibody is eluted immediately with4M Guanidine-HCl (pH 8.0) and the antigen matrix is washed with PBS. Theeluate is collected and measured (A₂₈₀). The elution efficiency of 4Mguanidine-HCl is functionally defined as 100%; there is no antibodyremaining on the column that can be further eluted with 4Mguanidine-HCl. Elution efficiencies calculated for other eluents (seebelow) are relative efficiencies using 4M guanidine-HCl as 100%.

The same antigen matrix is reacted with the same amount of PEG-purifiedchicken antibody and washed as discussed above. Bound chicken antibodyis eluted immediately with 2M Guanidine-HCl (pH 8.0) and the antigenmatrix is washed with PBS. The eluate is collected and measured (A₂₈₀).Relative elution efficiency is calculated as the total A₂₈₀ unitscollected here divided by the total A₂₈₀ units collected for 4Mguanidine-HCl.

The same antigen matrix is reacted a third time with the same amount ofPEG-purified chicken antibody and washed as above. Bound chickenantibody is immediately eluted with 8M Urea (pH 8.0) and the antigenmatrix is washed with PBS. The eluate is collected and measured (A₂₈₀).Relative elution efficiency is calculated as above.

The same antigen matrix is reacted a fourth time with the same amount ofPEG-purified chicken antibody and washed as above. Bound chickenantibody is immediately eluted with 4M Urea (pH 8.0) and the antigenmatrix is washed with PBS. The eluate is collected and measured (A₂₈₀).Relative elution efficiency is calculated as above.

The same antigen matrix is reacted a fifth time with the same amount ofPEG-purified chicken antibody and washed as above. Bound chickenantibody is immediately eluted with 0.5M diethylamine (pH 11.5) and theantigen matrix is washed with PBS. The eluate is collected and measured(A₂₈₀). Relative elution efficiency is calculated as the percent ofmaximum yield (see above). The efficiency of elution for the fiveeluents is shown in Table 2. It can be seen that 4M Urea and 0.5Mdiethylamine are poor eluents; they fail to remove all the bound chickenantibody. The other three eluents remove 90% or more of the boundantibody.

                  TABLE 2                                                         ______________________________________                                        Efficiency of Eluents                                                         Eluent              Efficiency                                                ______________________________________                                        4      M Guanidine-HCl, pH 8.0                                                                        100                                                   2      M Guanidine-HCl, pH 8.0                                                                        90                                                    8      M Urea, pH 8.0   100                                                   4      M Urea, pH 8.0   65                                                    0.5    M diethylamine, pH 11.5                                                                        38                                                    ______________________________________                                    

EXAMPLE 13 Elution of Non-Mammalian Antivenom Antibodies from anAldehyde-Activated Agarose Venom Antigen Matrix Using Guanidine

In this example, chicken antibody was eluted with 4M guanidine-HCl (pH8.0) from an antigen matrix made up with resin I. Approximately 10 mg C.atrox venom was coupled per ml of CNBr-activated Sepharose 4B as inExample 6 above. 100 ml of PEG-purified 355 (see Example 2) was loadedon a 5 ml antigen matrix at a flow rate of 1 ml per minute. The flowthrough ("355 flow through") was collected and the antigen matrix waswashed successively with several bed volumes of PBS, BBS-Tween (0.1Mboric acid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3),and PBS until the effluent was free of protein (A₂₈₀) Bound chickenantibody was eluted immediately with 4M guanidine-HCl (pH 8.0) and theantigen matrix was washed with PBS. The eluate ("Resin I purified 355")was collected and measured (A₂₈₀) and found to contain 125 μ g ofantibody per ml of PEG-purified 355 applied.

EXAMPLE 14 Elution of Non-Mammalian Antivenom Antibodies from anAldehyde-Activated Agarose Venom Antigen Matrix with a Non-DenaturingEluent

In this example, chicken antibody was eluted from an aldehyde-activatedresin with 4M guanidine-HCl (pH 8.0). 5 mg C. atrox venom was coupledper ml of ULTROGEL AcA 22 as in Example 7 above. 5 mls of PEG-purifiedPool 4 (9 mg/ml total protein) antibody was loaded on a 3 ml antigenmatrix at a flow rate of 1 ml per minute. The flow through ("Pool4/ultro flow through") and the antigen matrix was washed successivelywith several bed volumes of PBS, BBS-Tween (0.1M boric acid, 0.025Msodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), and PBS until theeffluent was free of protein (A₂₈₀) Bound chicken antibody wasimmediately eluted with 4M guanidine-HCl (pH 8.0) and the antigen matrixwas washed with PBS. The eluate ("ultro-purified Pool 4") was collectedand measured (A₂₈₀) and found to contain 742 μg of antibody per ml ofPEG-purified Pool 4 applied.

EXAMPLE 15 Increasing the Elution Efficiency of a Non-Denaturing Eluentby Increasing Column Residence Time

In this example, the elution efficiency of a recently developed elutionmedium on an aldehyde-activated resin was demonstrated. 10 mg C. atroxvenom was coupled per ml of ACTIGEL as in Example 8 to make the antigenmatrix. 10 mls of PEG-purified 355 (see Example 2) was loaded on a 5 mlantigen matrix at a flow rate of 1 ml per minute. The flow through ("355flow through") was collected and the antigen matrix was washedsuccessively with several bed volumes of PBS, BBS-Tween (0.1M boricacid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), andPBS until the effluent was free of protein (A₂₈₀). Bound chickenantibody was eluted with ACTISEP Elution Medium (Sterogene) according tothe manufacturer's instructions, i.e. eluent was applied in a mannersuch that it was in contact with the antigen matrix ("residence time")for 30 minutes. The formulation for ACTISEP is provided in U.S. patentappplication Ser. No. 06/197,714, which is hereby incorporated byreference. The eluate ("actigel/actisep-purified 355") was collected andmeasured (A₂₈₀), and the antigen matrix was washed with TBS. Remainingantibody on the antigen matrix was eluted with 4M guanidine-HCl. Thiseluate ("actigel guano purified 355" was collected and measured (A₂₈₀),and the antigen matrix was then washed with PBS. Elution efficiency wascalculated as the percent of the total antibody eluted by ACTISEP andwas found to be 26% (400 μg total protein). The remaining 74% (1.5 mgtotal protein) was eluted by quanidine.

EXAMPLE 16 Further Increasing the Elution Efficiency of a Non-DenaturingEluent

In this example, the elution efficiency of ACTISEP on an activatedaldehyde resin was optimized by a modified protocol; the residence timewas increased to 2 hours (Assay 1) and then to 2 hours and 45 minutes(Assay 2).

Assay 1

The cocktail matrix of Example 11 (above) was used to immunoaffinitypurify 10 mls of PEG-purified 355 (see Example 2). The antibody wasapplied as in Example 15 except that the residence time was increased to90 minutes (time 0 is just before the eluent is detectable in theeffluent of the column). The eluate was collected and measured (A₂₈₀),the antigen matrix was stripped of remaining antibody with 4Mguanidine-HCl, and this stripped antibody was collected and measured(A₂₈₀). Elution efficiency was calculated as in Example 15 and found tobe 47%.

Assay 2

The C. atrox matrix of Example 8 was used to immunoaffinity purify 10mls of PEG-purified Pool 2 (see Example 4). The antibody was applied asin Example 15 except that the residence time was increased to 2 hoursand 45 minutes (time 0 is just before the eluent is detectable in theeffluent of the column). The eluate was collected and measured (A₂₈₀),the antigen matrix was stripped of remaining antibody with 4Mguanidine-HCl, and this stripped antibody was collected and measured(A₂₈₀). Elution efficiency was calculated as in Example 15 and found tobe 73%.

The results of this example are shown in FIG. 8 (the results of Example15 are plotted in FIG. 8 for purposes of comparison). The efficiency ofantibody elution was increased to 47% with 90 minutes of residence timeand to 73% with 2 hours and 45 minutes of residence time.

EXAMPLE 17 Optimization of Elution Efficiency

In this example, the elution efficiency of ACTISEP on an activatedaldehyde resin was optimized by a further modified protocol. 10 mg C.atrox venom was coupled per ml of ACTIGEL to make an antigen matrix asin Example 8 above. Affinity purification was carried out as in FIG. 9.25 mls of PEG-purified Pool 2 (see Example 4) was loaded on the antigenmatrix at a flow rate of 1 ml per minute. The antigen matrix was washedand the bound antibody eluted with ACTISEP as in Example 15 except theresidence time was increased by stopping the flow of the column at thepoint where the peak of eluted protein concentration was reached. Asbefore, the antigen matrix was washed with TBS, the eluate was collectedand measured (A₂₈₀), the antigen matrix was stripped of remainingantibody with 4M guanidine-HCl, this stripped antibody was collected andmeasured (A₂₈₀), the antigen matrix was washed with buffer and stored at4° C. for later use.

The peaks in FIG. 9 are numbered to correspond to chicken antibody flowthrough (peak 1), non-specifically bound antibody (peak 2), ACTISEPeluted antibody (peak 3) (note ACTISEP baseline, i.e. absorbanceattributable to the eluent alone), and 4M guanidine-stripped antibody(peak 4). Importantly, by stopping the flow of the column at the pointwhere the peak of eluted protein concentration was reached, theefficiency of antibody elution could be further increased to 89%(efficiency calculated as in Example 14) for an ACTISEP elution of 128μgof antibody per ml of PEG-purified antibody applied. An additional 16μgof antibody (per ml pf PEG-purified antibody applied) was recovered withquanidine (for a total of 144 μg/ml of specific antibody per ml ofPEG-purified antibody applied).

EXAMPLE 18 The Increase in Titer of Antivenom antibody with FurtherImmunization

In this example, chicken antibody was quantitatively immunoaffinitypurified from an aldehyde-activated resin to show increasing antibodytiter with increasing immunization. 10 mg C. atrox venom was coupled perml of ACTIGEL A as in Example 8 above. The four, chicken #354,PEG-purified pools described in Example 4 were used. After each elution,the antigen matrix was washed with TBS, the eluate was collected andmeasured (A₂₈₀), the antigen matrix was stripped of remaining antibodywith 4M guanidine-HCl, this stripped antibody was collected and measuredA₂₈₀, the antigen matrix was washed and a new pool of chicken antibodywas loaded to the antigen matrix.

                  TABLE 3                                                         ______________________________________                                        Antivenom Antibody Titers                                                     #354 Pool   Titer (μg Ab/ml egg yolk)                                      ______________________________________                                        1            98                                                               2           144                                                               3           600                                                               4           905                                                               ______________________________________                                    

Each pool (5-20 mls) was loaded on the same 5 ml antigen matrix at aflow rate of 1 ml per min, and washed successively with several bedvolumes of PBS, BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1MNaCl, 0.1% (v/v) Tween 20 pH 8.3), and PBS until the effluent was freeof protein (A₂₈₀) Bound chicken antibody was eluted immediately with 4Mguanidine-HCl (pH 8.0) and the antigen matrix was washed with PBS. Theeluates were collected and measured (A₂₈₀) for amounts of specificantibody. The results are shown Table 3. The results demonstrate thatthe C. atrox-specific antibody titer increased nearly ten-fold over aperiod of approimately 60 days due to further immunizations.

EXAMPLE 19 Purity of Mammalian and Non-Mammalian AntivenomsImmunoaffinity Purified on an Aldehyde-Activated Whole Venom AntigenMatrix

In this example, the purity of antivenoms before and afterimmunoaffinity purification was demonstrated. 10 mg C-atrox venom wascoupled per ml of ACTIGEL A as in Example 8. 2 mls of unpurified horseantivenom (Wyeth; log #M878035) containing 210 mg/ml (total 420 mg) wereapplied to a 5 ml antigen matrix, the flow-through was washed throughthe column initially with PBS and saved for further analysis, the matrixwas then washed with BBS-Tween until the effluent was substantially freeof protein (A₂₈₀) and then with PBS. Bound antibody was elutedimmediately with 4M guanidine, collected and measured (A₂₈₀) aftercomplete dialysis. The antigen matrix was then re-equilibrated with PBS.This eluate contained 19.7 A₂₈₀ units of antibody.

The flow-through was then re-applied to the 5 ml C.atrox venom antigenmatrix and washed and eluted as described above in order to affinitypurify any antibody not isolated in the first pass above. Theguanidine-HCl eluate from the second pass contained only 3.4 A₂₈₀ unitsof antibody, indicating that approximately 85% of the C.atrox specificantibody was purified in the first pass. Importantly, the 23.1 A₂₈₀units (16.5 mg) of total antibody purified from both passes representsonly 3.7% (16.5 mg/420 mg) of the total A₂₈₀ units of protein present inthe crude horse antivenom, indicating that 95% or more of the proteinpresent in the antivenom does not react with C.atrox venom. This is incontrast with the pool #4 affinity purified chicken anti-C.atroxdescribed in Example 18 where 905 μg/ml of a 9 mg/ml PEG prep or 10% ofthe total protein was C.atrox-specific antibody. Because of the higherconcentration of antivenom antibodies and increased protein homogeneity(see below) of the chicken IgY, we contemplate that this antivenom,without affinity purification, is useful for passive immunization (aswell as in vitro analytical work).

To examine the composition of the crude and affinity purifiedantivenoms, analytical SDS-PAGE was performed on antivenoms at differentstages of purification. A fresh 2 ml sample of crude horse antivenom(Wyeth; lot #M878035) was applied to the same 5 ml C.atrox venom antigenmatrix described above and the column washed with PBS, BBS-Tween, andTBS until the effluent was substantially free of protein (A₂₈₀). Boundantibody was eluted with ACTISEP using the optimized protocol in Example17 and collected and measured after complete dialysis. The antigenmatrix was then washed with TBS and the remaining antibody elutedimmediately with 4M guanidine-HCl, collected and measured (A₂₈₀) aftercomplete dialysis. The antigen matrix was then re-equilibrated bywashing with PBS. Samples of the unpurified, flow-through, ACTISEP andguanidine fractions were retained for further analysis.

Samples from the affinity purification of 25 mls of Pool 2 described inExample 17 were analyzed along with the horse antivenom samples bySDS-PAGE on a 10% reducing gel (FIG. 10). Comparison of 100 μg of theapplied crude horse antiserum (Lane 1) with 100 μg of the crude horseantiserum flow through (Lane 2) revealed no detectable differences incomposition. However, 30 μg (Lane 3) and 150 μg (Lane 5) of theguanidine-eluted horse antibody and 30 μg of the ACTISEP-eluted horseantibody (Lane 4) all exhibited a pattern of fewer polypeptides; thepredominant polypeptide was found to be a 55,000 dalton band (size isestimated from molecular weight markers in Lane 7) corresponding to therelative mobility of the heavy chain of horse immunoglobulin. None ofthe higher molecular weight polypeptides present in the crude antivenom(Lanes 1 and 2) were found in the eluates (Lanes 3, 4 and 5), indicatingthat these high molecular weight proteins are not immunoglobulin and areremoved during affinity purification. Densito-metric scanning of gellane 1 indicated that no more than 37% of the total protein present inthe crude Wyeth antivenom was immunoglobulin (data not shown). Since3.7% of the total protein is venom-reactive (see above), it follows that10% (3.7%/37%) of the total horse immunoglobulin is venom-reactive.

Similarly, 30 μg of the PEG-purified, chicken antibody flow through(Lane 9) contains high molecular weight polypeptides that are notrecovered in the guanidine (Lane 10) and ACTISEP (Lane 11) eluates. Thebands associated with these eluates (Lanes 10 and I1) correspond to thepolypeptides of a commercial (Cappel) sample of pure chickenimmunoglobulin (Lane 6). (The proteins from unpurified chicken yolks areshown in Lane 8). These results demonstrate that: i) the PEG-purifiedchicken antivenom (Lane 9) is less heterogenous than the crude horseantivenom (Lane 1), in that far fewer non-immunoglobulin proteins arepresent in the PEG-purified chicken IgY preparation (greater than 90% ofthe total PEG-purified protein is immunoglobulin and, given that 10% ofthe protein is venom reactive, it follows that 11% (10%/90%) of theimmunoglobulin is venom-reactive); ii) the affinity purification removessubstantially all of the non-immunoglobulin protein from the crudeantivenoms (greater than 99% of the protein in these preparations isimmunoglobulin); and iii) both the ACTISEP and guanidine eluatescontaining the horse and chicken antivenoms are essentially pure(greater than 99%) antigen-specific immunoglobulin. Table 4 summarizesthe immunoglobulin content, purity and reactivity of the horse andchicken antivenoms at different stages of purification. Note that beforeaffinity purification (see "crude horse" and "PEG IgY"), less than 50%of the antivenom immunoglobulin ("Ig") is venom reactive.

                  TABLE 4                                                         ______________________________________                                        Immunoglobulin Content, Purity & Reactivity                                   Antivenom                                                                              % Ig     % reactive protein                                                                          % reactive Ig                                 ______________________________________                                        Crude Horse                                                                             37      3.7           10                                            AP Horse >99      >99           >99                                           PEG IgY  >90      10            11                                            AP IgY   >99      >99           >99                                           ______________________________________                                    

After affinity purification ("AP"), greater than 50% of the antivenomimmunoglobulin ("Ig") is venom-reactive.

EXAMPLE 20 Spectrum of Reactivity of Horse Antivenom before and afterAffinity Purification on a C. atrox Antigen Matrix

In this example, the ability of a C. atrox antigen matrix to bind andpurify the spectrum of antibodies present in a crude polyvalent horseantivenom was examined. 2 ml of Wyeth Polyvalent Crotalid antivenom (lot#M878035), raised against C. atrox, C. adamanteus, B. atrox, and C.durissus terrificus venoms, was applied to a 5 ml C. atrox Actigel Aantigen matrix prepared as described in Example 8. The flow-through wascollected, the column washed, and antibody eluted as described inExample 17. The flow-through fraction was applied to two more C.atrox-Actigel A antigen matrices in succession in order to remove asmuch C. atrox reactive antibody as possible. The total amount of C.atrox reactive antibody, calculated from the A₂₈₀ of all three columneluates, was 7.5 mg per ml of this lot of Wyeth antivenom.

To assess the spectrum of activity of the purified C. atrox-reactiveantibody, and to compare it with the activity of both the crudeantivenom and the non-C. atrox reactive fraction of the antivenom thatflowed through all three C. atrox matrices, we examined antibodyreactivity by ELISA using the four original venoms used for immunizationas antigens (see above). 96-well Nunc Plates were coated overnight at 4°C. in a humidified chamber with 200 μl/well of the appropriate venomdissolved in PBS at 5 μg/ml. The venoms used in this example were C.atrox, C. adamanteus, C. durissus terrificus, and B. atrox (Sigma). Thenext day the wells were blocked with PBS containing 0.1% bovine serumalbumin for 2 hours at room temperature. Antibodies to be tested forbinding to each of the four venoms were diluted in PBS containing 2%(v/v) normal goat serum (GIBCO). In order to directly compare the crudeWyeth antivenom, the antigen matrix flow-through fraction, and theaffinity purified anti-C. atrox with respect to their reactivities, theantibodies were diluted in such a way as to normalize i) the volume ofthe flow-through fraction with the volume of the crude Wyeth antivenomstarting material, and ii) the concentration of C. atrox-specificantibodies in the crude antivenom, i.e., since the crude antivenomcontain 7.5 mg/ml of C. atrox-specific antibody, to compare theunpurified crude antivenom to a set concentration of purified antibodydilutions were made of the crude material such that the concentrationsof C. atrox-specific antibody were the same (e.g., 2.5 μ g/ml ofspecific antibody is equivalent to a 1:3000 dilution of crudeantivenom).

200 μl/well of four 3-fold serial dilutions of each antibody and anormal unimmunized horse serum control (Sigma) were incubated for 2hours at room temperature. The plates were then washed three times withBBS containing 0.1% Tween 20, twice with PBS-Tween 20, and twice withPBS. Alkaline phosphatase-conjugated goat anti-horse IgG (FisherBiotech) was diluted 1:500 in PBS containing 2% (v/v) normal goat serum,added to the plates, and incubated 2 hours at room temperature. Theplates were washed as before, except Tris-buffered saline wassubstituted for the last wash, and p-nitrophenyl phophate was added andthe hydrolysis of the substrate measured at 410 nm as described inExample 2.

                  TABLE 5                                                         ______________________________________                                        Reactivity of Purified Horse Antivenom                                                 % of initial reactivity with                                                         C.                 C. durissus                                         C. atrox                                                                             adamanteus                                                                              B. atrox terrificus                                 ______________________________________                                        Starting Material                                                                        100      100       100    100                                      (crude Wyeth)                                                                 C. atrox Flow-                                                                           11        7        21     49                                       through                                                                       C. atrox affinity                                                                        88       97        85     40                                       purified                                                                      ______________________________________                                    

The ELISA results of the relative reactivity of the crude horseantivenom, the C. atrox antigen matrix flow-through, and the affinitypurified anti-C. atrox antibody at a concentration of antibody that fellwithin the linear range of the ELISA (the normal horse serum controlvalues have been subtracted) on each of the four venoms are shown inTable 5. The results show that affinity purification of this antivenomwith the C. atrox antigen matrix recovers the majority of the antibodyactivity against three of the original venoms used for immunization, C.atrox, c. adamanteus, and B. atrox, indicating that these three venomscontain many similar antigens. However, the fourth venom, C. durissusterrificus, is not as strongly reactive with the C. atrox purifiedantibody. In fact, the majority of the C. durissus terrificus reactivityby this assay still remains in the flow-through fraction from the C.atrox antigen matrix. These examples demonstrate the existence of atleast two antibody populations in the crude Wyeth antivenom, one that ispolyvalent and reactive with C. atrox and the other three venoms, andone that is C. durissus terrificus reactive but not C. atrox-reactive(C. durissus terrificus monovalent).

EXAMPLE 21 Identification and Purification of Two Monovalent AntivenomAntibody Subpopulations and One Polyvalent Subpopulation in a HorseAntivenom by Sequential Imunoaffinity Chromatography

In this example, the reactivity and cross-reactivity of differentsubpopulations of antibodies derived from the Wyeth antivenom wereexamined in order to determine their valency. In addition, a means isdemonstrated for purifying two or more monovalent subpopulations ofantivenom antibodies by affinity chromatography of whole venom oversuccessive antigen matrices composed of antigenically distinct venoms.

In Example 20, it was demonstrated that the antivenom fraction that didnot bind to the C. atrox antigen matrices contained a large fraction ofthe original C.durrisus terrificus venom reactivity of the crude venom.To purify, quantitate, and analyze this subpopulation of antibodies,1/7th of the flow-through fraction from three successive C. atroxantigen matrices was applied to a 3 ml C. durissus terrificus Actigel Aantigen matrix prepared as described in Example 9. The flow-throughfraction was saved, the matrix washed, and antibody eluted with 4Mguanidine-HCl, collected, and measured (A₂₈₀) as described in Example13. The results showed that there exists approximately 1.2 mg of C.durissus-reactive antibody per ml of Wyeth antivenom that will not bindto a C. atrox antigen matrix. This is the C. durissusterrificus--specific monovalent antibody subpopulation. To examine theefficiency of the C. durissus terrificus antigen matrix at isolating theC. durissus terrificus--reactive antibody and to examine the activity ofthe affinity purified anti- C. durissus terrificus antibody, an ELISAwas performed to assess the reactivity and cross-reactivity of theoriginal Wyeth antivenom, the flow-through fraction of the C. atroxmatrices before application to the C. durissus terrificus matrix, theflow-through fraction after application to the C. durissus terrificusmatrix, the affinity purified anti-C. durissus terrificus antibody, andthe affinity purified anti-C. atrox antibody from Example 20. All fourof the original immunizing venom antigens were coated on Nuncimmunoplates and the antigen binding activity of various dilutions ofthe different antivenom antibody fraction assessed exactly as describedin Example 20. In order to compare the antigen-binding activities ofdifferent fractions, values were normalized to either the originalstarting volume of antibody, or to the original starting concentrationof C. atrox-reactive antibody. The ELISA results of the relativereactivity of the different preparations at a specific antibodyconcentration that fell within the linear range of the assay are shownin Table 6. By passing the crude Wyeth antivenom sequentially over theC. atrox antigen matrix followed by the C. durissus terrificus antigenmatrix, one recovers in the sum of the two purified antibodies more than66% of the crude antivenom's initial reactivity with all four venoms.

                  TABLE 6                                                         ______________________________________                                        Retention of the Spectrum of Reactivity of                                    a Horse Antivenom by Sequential Immunoaffinity                                Chromatography                                                                             C.   C.        B.     C. durissus                                             atrox                                                                              adamanteus                                                                              atrox  terrificus                                 ______________________________________                                        Crude Wyeth AV 100    100       100  100                                      C. atrox flow-through                                                                         8     10        22   55                                       before C. durissus                                                            matrix                                                                        C. atrox/C. durissus                                                                          8      8        20   10                                       terrificus matrices                                                           flow-through                                                                  Affinity purified                                                                             1     <1        <1   38                                       anti-C. durissus                                                              terrificus                                                                    Affinity purified                                                                            84     70        66   38                                       anti-C. atrox (Ex. 20)                                                        Sum of two affinity                                                                          85     70        66   76                                       purified antibodies                                                           ______________________________________                                    

The important function of the second matrix was to purify the vastmajority of the C. durissus terrificus--reactive antibody that would notbind to the first (C. atrox) matrix.

The anti-C. atrox-reactive antibody population that bound to the C.atrox matrix was further fractionated by applying 3.2 A₂₈₀ units ofaffinity purified antibody prepared as described in Example 20 to thesame C. durissus terrificus Actigel A antigen matrix described above.Two antibody subpopulations were obtained. The first was theflow-through fraction that did not bind to the C. durissus terrificusmatrix. The second was eluted from the column with 4M guanidine-HClpH8.0 after washing the matrix with PBS, BBS-Tween and PBS as in Example13. The eluate was collected, dialyzed against PBS, and measured (A₂₈₀).The results showed that 0.63 of the original A₂₈₀ units applied werebound to and eluted from the antigen matrix, thus 20% of the C.atrox-reactive antibody isolated on a C. atrox matrix is also reactivewith C. durissus terrificus. This antibody comprises one polyvalent(with respect to C. atrox and C. durissus terrificus) antibodysubpopulation of the original antivenom.

The unbound flow-through fraction constitutes the C. atrox-reactive, C.durissus terrificus non-reactive antibody subpopulation of the originalantivenom, or the C. atrox-specific monovalent subpopulation.

To confirm the venom binding reactivity of the three different antibodysubpopulations identified in this example, an ELISA was performedexactly as described above and the C. atrox-reactivity and C. durissusterrificus-reactivity of the C. durissus terrificus-specific monovalentantibody subpopulation, the C. atrox-specific monovalent antibodysubpopulation, and the C. atrox/C. durissus-specific polyvalent antibodysubpopulation compared as the two antigens. The results are shown inFIGS. 11A, 11B and 11C for the three subpopulations, respectively. Theresults demonstrate that the monovalent subpopulations are stronglyreactive only with their respective single venoms while the polyvalentsubpopulation is strongly reactive with both venoms.

EXAMPLE 22

Purification of the broad spectrum of venom-reactive antibodies presentin a crude mammalian antivenom in one step using a cocktail matrix.

An alternative to purifying antivenom antibodies on successive antigenmatrices was demonstrated. In this example, 2.5 ml. of a C.atrox-Actigel A antigen matrix prepared as described in Example 8 and2.5 ml. of a C. durissus terrificus antigen matrix prepared as describedin Example 9 were mixed together to form one combination antigen matrix.0.25 ml of Wyeth antivenom was diluted with 10 mls of PBS and applied tothe combination antigen matrix, the flow-through fraction was saved andthe matrix was washed with PBS, BBS-Tween, and TBS and specific antibodyeluted with Actisep elution medium as described in Example 17 The eluatewas collected, dialyzed against PBS, and measured (A₂₈₀). The resultsshowed that approximately 2.2 mg of specific antibody was eluted fromthe matrix, which corresponds to a specific antibody titer of 8.8 mg.per ml. of Wyeth antivenom. This correlates well with previousdeterminations of 7.5 mg. of C.atrox-and 1.2 mg. of C. durissusterrificus reactive antibody (7.5+1.2=8.7 which is very close to 8.8)per ml of Wyeth antivenom (see Examples 19 and 21).

The unbound and eluted fractions were assayed by ELISA as described inExample 20 in order to determine the spectrum of reactivity of thechromatographic fractions before and after purification. To calculatethe percent of the initial reactivity on each of the four venoms, valueswere normalized to either the original starting volume of antivenom, theoriginal concentration of C-durissus terrificus-reactive antibody, orthe original concentration of C. atrox-reactive antibody. The reactivityof different samples at a concentration of antibody that fell within thelinear range of the ELISA assay were compared and are shown in Table 7.

These results demonstrate that antibodies reactive with all four venomscan be substantially purified by application to a combination antigenmatrix composed of the two most antigenically distinct venoms used forimmunization. Importantly, the spectrum of reactivity of the antivenomantibody purified in a single step in this example is comparable to thesum of reactivities of the C. atrox- and C. durissus terrificus reactingantibodies that were sequentially purified in Example 21.

                  TABLE 7                                                         ______________________________________                                        Reactivity of Cocktail-Matrix-Purified Antivenom                                           C.   C.        B.     C. durissus                                             atrox                                                                              adamanteus                                                                              atrox  terrificus                                 ______________________________________                                        Crude Wyeth AV 100    100       100  100                                      Combination matrix                                                                           11     16        20   12                                       flow-through                                                                  Affinity purified                                                                            73     77        69   71                                       antibody from                                                                 combination matrix                                                            Sum of affinity                                                                              85     70        66   76                                       purified anti-C. atrox                                                        and C. durissus terrificus                                                    from Example 21                                                               ______________________________________                                    

EXAMPLE 23

Purification of avian antibodies by sequential immunoaffinitychromatography over individual antigen matrices and in one step using acocktail matrix.

In this example, chicken antibody was eluted from a cocktail antigenmatrix and compared with antibody eluted from single-antigen matrices. 8mg C. atrox venom was coupled per ml of ACTIGEL A as in Example 8. 8 mgC. adamanteus venom was coupled per ml of ACTIGEL A as in Example 9.Pool 4 PEG-prep (see Example 4) from chicken #354 was used in threeseparate assays.

Assay 1

2 mls of PEG-purified 127 (containing approximately 3.0 A₂₈₀ units ofspecific antibody) was loaded on a 2 ml C. atrox antigen matrix(approximately 16 mg of total venom protein) at a flow rate of 1 ml perminute. The flow through was carefully collected and the C. atroxantigen matrix was washed successively with several bed volumes of PBS,BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v)Tween 20 pH 8.3), and PBS until the effluent was free of protein (A₂₈₀)Bound chicken antibody was eluted ("eluate A") immediately with 4Mguanidine-HCl (pH 8.0) and the C. atrox antigen matrix was washed withPBS.

The flow through was thereafter loaded on the C. adamanteus antigenmatrix (2 mls; approximately 16 mg of total venom protein) at a flowrate of 1 ml per minute. The C. adamanteus antigen matrix was washedsuccessively with several bed volumes of PBS, BBS-Tween (0.1M boricacid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), andPBS until the effluent was free of protein (A₂₈₀). Bound chickenantibody was eluted ("eluate B") immediately with 4M guanidine-HCl (pH8.0) and the antigen matrix was washed with PBS. Eluates A and B weremeasured (A₂₈₀) and found to contain the following amounts of specificantibody:

    ______________________________________                                        Eluate      Protein (A.sub.280)                                               ______________________________________                                        A           2.86                                                              B           0.11                                                              ______________________________________                                    

Thus, when antibody is passed sequentially over a C. atrox antigenmatrix and a C. adamanteus antigen matrix, the vast majority ofvenom-specific antibody is pulled out by the C. atrox antigen matrix.

Assay 2

2 mls of Pool 4 PEG-prep (approximately 3.0 A₂₈₀ units of specificantibody) was loaded on a 2 ml C. adamanteus antigen matrix(approximately 16 mg of total venom protein) at a flow rate of 1 ml perminute. The flow through was carefully collected and the C. adamanteusantigen matrix was washed successively with several bed volumes of PBS,BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v)Tween 20 pH 8.3), and PBS until the effluent was free of protein (A₂₈₀).Bound chicken antibody was eluted ("eluate C") immediately with 4Mguanidine-HCl (pH 8.0) and the C. adamanteus antigen matrix was washedwith PBS.

The flow through was thereafter carefully loaded on the C. atrox antigenmatrix (2 mls; approximately 16 mg of total venom protein) at a flowrate of 1 ml per minute. The C. atrox antigen matrix was washedsuccessively with several bed volumes of PBS, BBS-Tween (0.1M boricacid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), andPBS until the effluent was free of protein (A₂₈₀) Bound chicken antibodywas eluted ("eluate D") immediately with 4M guanidine-HCl (pH 8.0) andthe C. atrox antigen matrix was washed with PBS. Eluates C and D weremeasured (A₂₈₀) and found to contain the following amounts of specificantibody:

    ______________________________________                                        Eluate      Protein (A.sub.280)                                               ______________________________________                                        C           1.98                                                              D           0.92                                                              ______________________________________                                    

Thus, when antibody is passed sequentially over a C. adamanteus antigenmatrix and a C. atrox antigen matrix, a significant portion ofvenom-specific antibody is left behind by the C. adamanteus antigenmatrix.

Assay 3

2 mls of the C. atrox antigen matrix was mixed with 2 mls of the C.adamanteus antigen matrix to make a 4 ml cocktail antigen matrix. 2 mlsof Pool 4 PEG-prep (approximately 3.0 A₂₈₀ units of specific antibody)was loaded on the cocktail antigen matrix at a flow rate of 1 ml perminute. The flow through ("first flow through") was carefully collectedand the cocktail antigen matrix was washed successively with several bedvolumes of PBS, BBS-Tween (0.1M boric acid, 0.025M sodium borate, 1MNaCl, 0.1% (v/v) Tween 20 pH 8.3), and PBS until the effluent was freeof protein (A₂₈₀). Bound chicken antibody was eluted ("eluate E")immediately with 4M guanidine-HCl (pH 8.0) and the cocktail antigenmatrix was washed with PBS.

The first flow through was thereafter loaded on a C. atrox antigenmatrix (2 mls; approximately 16 mg of total venom protein) at a flowrate of 1 ml per minute. Again, the flow through ("second flow through")was carefully collected. The C. atrox antigen matrix was washedsuccessively with several bed volumes of PBS, BBS-Tween (0.1M boricacid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), andPBS until the effluent was free of protein (A₂₈₀). Bound chickenantibody was eluted ("eluate F") immediately with 4M guanidine-HCl (pH8.0) and the C. atrox antigen matrix was washed with PBS.

The second flow through was thereafter loaded on a C. adamanteus antigenmatrix (2 mls; approximately 16 mg of total venom protein) at a flowrate of 1 ml per minute. The C. adamanteus antigen matrix was washedsuccessively with several bed volumes of PBS, BBS-Tween (0.1M boricacid, 0.025M sodium borate, 1M NaCl, 0.1% (v/v) Tween 20 pH 8.3), andPBS until the effluent was free of protein (A₂₈₀). Bound chickenantibody was eluted ("eluate G") immediately with 4M guanidine-HCl (pH8.0) and the C. adamanteus antigen matrix was washed with PBS.

Eluates E, F and G were measured (A₂₈₀) and found to contain thefollowing amounts of specific antibody:

    ______________________________________                                        Eluate      Protein (A.sub.280)                                               ______________________________________                                        E           2.77                                                              F           0.17                                                              G           0.06                                                              ______________________________________                                    

Thus, the cocktail antigen matrix pulls out the vast majority ofvenom-specific antibody in the chicken antibody preparation.

The results of all three assays demonstrate that the C. atrox and C.adamanteus venoms are immunologically similar (consistent with theresults of Examples 20-22 obtained with a mammalian antivenom). Becauseof this immunochemical similarity (and the fact that boosting venomswere only C. atrox and C. adamanteus), it was expected that eitherantigen matrix alone would be efficient at retaining the spectrum ofantibody reactivity of the unpurified avian antivenom. Surprisingly, C.adamanteus is not efficient in this respect. For this reason, allfurther tests of chicken anti-Crotalid antivenom was performed withantibody affinity purified on a C. atrox antigen matrix.

EXAMPLE 24

Recyclability of an aldehyde-activated agarose venom antigen matrix.

In this example, the stability and recyclability of analdehyde-activated venom antigen matrix was demonstrated. C. atrox venomwas dissolved at 10 mg 1 ml in PBS and coupled to Actigel A (Sterogene)as described in Example 8. 2 ml of crude horse antiserum (lot #M878035)was loaded on this antigen matrix (5 ml column) at 1 ml per minute tobegin each purification cycle (one complete cycle is described inExample 15; see FIG. 6). The antigen matrix was subjected to repeatedcycles of antivenom application, matrix washing, and specific antibodyelution. The amount of antibody purified from was quantitated at fourdifferent time points over a 260 day period. The results are shown inTable 8. The mean A₂₈₀ units recovered were 20.4 with approximately 15%variation. The results show that no significant loss of capacityoccurred after eleven complete column cycles and 254 days of columnlife. Thus, the aldehyde-activated venom antigen matrix is stable andrecyclable.

                  TABLE 8                                                         ______________________________________                                        Matrix Recyclability                                                          Day      # of Previous Cycles                                                                         Total A.sub.280 Units                                 ______________________________________                                        6        2              23.1                                                  7        4              17.4                                                  62       9              19.0                                                  254      11             22.1                                                  ______________________________________                                    

EXAMPLE 25

Antigen-binding activity of guanidine-eluted antivenom antibodies from aCNBr-agarose venom antigen matrix.

The quality of venom-specific antibody eluted from an antigen matrixusing Resin I was determined by reactivity in an ELISA. 96-well Nunc(VWR Scientific, San Francisco) ImmunoPlates were coated overnight at 4°C. in a humidified chamber with 200 μl/well of C. atrox venom at aconcentration of 2 μg/ml. The next day the wells were blocked with PBScontaining 0.1% bovine serum albumin (BSA) for 2 hours at roomtemperature. To perform the ELISA, i) unimmunized whole chicken serum,ii) PEG-purified 355, iii) 355 flow through, and iv) Resin I purified355 (see Example 13 for description of 355 preparation) wereappropriately diluted (PEG-purified 355 and 355 flow through werediluted according to the specific antibody concentration of Resin Ipurified 355) in PBS (containing 0.1% BSA) and added to the wells induplicate. The plates were incubated for 2 hours at room temperature.The plates were then washed three times with BBS (0.1M boric acid,0.025M sodium borate, 1M NaCl, pH 8.3) containing 0.1% Tween 20, twicewith PBS containing 0.1% Tween, and twice with just PBS. Alkalinephosphatase-conjugated rabbit anti-chick IgG (Fisher) was diluted 1:500in PBS containing 0.1% BSA, added to the plates, and incubated 2 hoursat room temperature. The plates were washed as before, exceptTris-buffered saline, pH 7.2, was substituted for PBS in the last wash,and p-nitrophenyl phosphate (Sigma) was added at 1 mg/ml in 0.05M Na₂CO₃ pH 9.5, 10 mM MgCl₂. The plates were then evaluated quantitativelyby reading at 410 nm on a Dynatech MR300Micro ELISA reader approximately30 minutes after the substrate was added.

The results are shown in FIG. 12. Appropriately, no reactivity is seenwith IgY from an unimmunized bird (UB). Similarly, very littlereactivity is seen in the 355 flow through (FT). The PEG-purified 355 ishighly reactive (PEG). The Resin I purified 355 (RP) is also reactivebut is found to be only approximately 50% as reactive as PEG-purified355 starting material.

EXAMPLE 26

Antigen-binding activity of antivenom antibodies eluted from differentvenom antigen matrices with different eluents.

The quality of venom-specific antibody eluted from two differentaldehyde-activated antigen matrices was determined by reactivity in anELISA. 96-well plates were coated with C. atrox venom and blocked as inExample 20. To perform the ELISA, i) PEG-purified Pool 4, ii)ultro-purified Pool 4 (see Example 14) and iii) actigel/actisep-purifiedPool 4 (see Example 17) were appropriately diluted (PEG-purified Pool 4was diluted according to the specific antibody concentration of purifiedPool 4) in PBS (containing 0.1% BSA) and added to the wells induplicate. The plates were incubated and washed, and alkalinephosphatase-conjugated rabbit anti-chick IgG was diluted, added to theplates, and incubated; the plates were washed, p-nitrophenyl phosphatewas added and the plates were read (see Example 20).

The results are shown in FIG. 13. The PEG-purified Pool 4 is highlyreactive (PEG). The actigel / actisep-purified Pool 4 (SEP) is alsohighly reactive; 75% or more of the reactivity of the starting materialis retained. The ultro-purified Pool 4 (TRO) is less reactive; onlyapproximately 50% of the reactivity of the starting material isretained.

EXAMPLE 27

Improved activity of antivenom antibodies when eluted withnon-denaturing eluents.

The quality of venom-specific antibody eluted from aldehyde-activatedantigen matrices with two different eluents was determined by reactivityin an ELISA. 96-well plates were coated with C. atrox venom and blockedas in Example 20. To perform the ELISA, i) actigel/actisep-purified 355and ii) actigel/guano-purified 355 (see Example 15) were appropriatelydiluted in PBS (containing 0.1% BSA) and added to the wells induplicate. The plates were incubated and washed, and alkalinephosphatase-conjugated rabbit anti-chick IgG was diluted, added to theplates, and incubated; the plates were washed, p-nitrophenyl phosphatewas added and the plates were read (see Example 20).

The results are shown in FIG. 14 and indicate that, when identicalantibody concentrations of antibody are compared, ACTISEP-elutedantibody is more reactive than guanidine-eluted antibody.

EXAMPLE 28

Reactivity of antivenom antibodies with individual venom componentsbefore and after affinity purification.

The nature of the reactivity of affinity-purified antivenom wasdemonstrated by western blot. 100 μg samples of Crotalus adamanteus,Crotalus atrox, and Agkistrodon piscivorus venom were dissolved in SDSreducing sample buffer and heated at 95° C. for 10 minutes as inExample 1. The samples were then separated on a 10-20% gradient reducingSDS-PAGE. One strip of the gel was stained with Coomassie Blue. Theproteins in the remaining portion of the gel were transferred tonitrocellulose, the nitrocellulose was temporarily stained to visualizethe lanes, destained, and blocked (see Example 1). The blot was cut intostrips and each strip incubated with the appropriate primary antibody.PEG-purified pool 3 of chicken #354 and affinity-purified pool 3 ofchicken #354 (see Example 18) were diluted in PBS (containing 1 mg/mlBSA) at a concentration of 2.5 μg/ml of specific antibody and added tothe appropriate strip for 2 hours at room temperature. The strips werewashed with 2 changes each of large volumes of PBS, BBS-tween and PBSsuccessively (10 min/wash). Goat anti-chicken IgG alkaline phosphataseconjugated secondary antibody (Fisher Biotech) was diluted 1:400 in PBScontaining 1 mg/ml BSA and incubated with the blot for 2 hours at roomtemperature. The blots were washed with 2 changes each of large volumesof PBS and BBS-Tween, followed by 1 change of PBS and 0.1M Tris-HCl, pH9.5. Blots were developed in freshly prepared alkaline phosphatasesubstrate buffer: 100 mg/ml Nitro-Blue Tetrazolium (Sigma), 50 mg/ml5-Bromo-4-Chloro-3-Indolyl Phosphate (Sigma), and 5 mM MgCl₂ in 50 mMNa₂ CO₃ pH 9.5.

The results are shown in FIG. 15. Lane numbers correspond to the venoms:Crotalus adamanteus was placed in Lane 1; Crotalus atrox, was placed inLane 2; Agkistrodon piscivorus was placed in Lane 3. The Coomassie Bluestrip (Strip a) show the proteins of the different venoms. Strip b wasblotted with PEG-purified pool 3. Strip c was blotted with theaffinity-purified pool 3. A comparison of Strips b and c shows that thetwo purified preps exhibit reactivities against the same individualcomponents of the three venoms, indicating that there has been nosignificant loss of antibody reactivity against any particularcomponents of the venom.

EXAMPLE 29 Reactivity and Crossreactivity of an Immunoaffinity PurifiedNon-Mammalian Antivenom with Immunizing and Non-Immunizing Venoms.

The crossreactivity of affinity-purified antivenoms was demonstrated bywestern blot. 100 μg venom samples from 14 different snakes weredissolved in sample buffer, heated and separated on a 10-20% gradientreducing SDS-PAGE. Protein transfer, staining, destaining, and blockingwere as in Example 28. The entire blot was incubated for 2 hours at roomtemperature with anti-C. atrox immunoaffinity-purified antibody (Pool#4) that was diluted in PBS (containing 1 mg/ml BSA) to a concentrationof 2.5 μg/ml. The blot was washed, alkaline phosphatase-conjugated, goatanti-chicken Ig (Fisher) secondary antibody was added, and the blot waswashed again and developed as in Example 28.

The results are shown in FIG. 16. Lane numbers correspond to the venoms:Lane 1, A. piscivorus; lane 2, A. contortrix: lane 3, A. rhodostoma:lane 4, B. atrox; lane 5, C. atrox; lane 6, C. adamanteus; lane 7, C.viridis viridis; lane 8, C. horridus horridus; lane 9, C. ruber; lane10, C. scutulatus: lane 11, C. durissus terrificus; lane 12, Viperarusselli: lane 13, Trimereserus elegans; lane 14, Echis carinatus.

The degree of crossreactivity reflected geographic and evolutionaryrelationships. The antivenom reacted most strongly against venom from C.horridus horridus (central and eastern U.S.), C. ruber (Mexico), and C.viridis viridis (Central U.S.); no reactivity was observed againstvenoms from C. durissus terrificus (South America), A. rhodostoma(Southeast Asia), and Trimereserus elegans (Asia). Significantly, theimmunoaffinity purified, anti-C. atrox antivenom reacted poorly withvenom from C. scutulatus, an extremely neurotoxic venom from asouthwestern United States rattlesnake. Since C. scutulatus was notpresent in the original immunizing cocktail, these results indicate thati) there is practically no anti-C. atrox antibody that is crossreactivewith C. scutulatus venom and ii) there may be very little crossreactiveantibody generated with the particular cocktail used for immunization.This underscores the importance of both the cocktail used on the antigenmatrix and the cocktail selected for immunization. This also illustratesthe utility of the antivenoms of the present invention in selectingappropriate cocktails.

EXAMPLE 30

In vivo neutralization of rattlesnake venom lethality by affinitypurified non-mammalian antivenom antibody.

In this example, the ability of antivenom immunoaffinity purified on aC. atrox antigen matrix to neutralize the lethal effect of C. atroxvenom in mice was demonstrated. To first establish the lethal dose of C.atrox venom in mice, whole venom was dissolved and diluted in saline(0.85% NaCl) to give different doses of venom per unit of mouse bodyweight. It was observed that 7 mg of C. atrox venom per kg of bodyweight was usually fatal within 24 hours when injectedintraperitoneally.

To determine the venom-neutralizing activity of the affinity purifiedantivenom antibodies, the Actisep eluted antibody from Pool 3 in Example18 was concentrated on a Centricon-30 concentration unit (Amicon;Bedford, Mass.) to 4 mg of antibody per ml of PBS. Identical amounts ofthis antibody and a control non-immune chicken antibody (Cappel) wereseparately mixed with a fixed amount of C. atrox venom in saline,incubated for one hour, and a 110-130 μl dose (the particular dosevaried according to individual body weights) of each mixture wasinjected into 9-10 live mice of between 25-34 grams in weight. The micewere observed for 24 hours and the results of the example are shown inTable 9. Thus, the affinity purified anti-C. atrox antibody exhibitedcomplete protection of the experimental mice from the lethal effects ofthe venom. The statistical significance of the effect of the antivenomantibody treated mice survival frequency was examined by Chi-squareanalysis and the value for p was determined to be <0.01. This is thefirst demonstration of in vivo neutralization of a venom by an avianantivenom.

                  TABLE 9                                                         ______________________________________                                        Neutralization of Venom In Vivo                                               Venom Dose Antibody Type and                                                                           Number of Number of                                  (mg/kg)    Dose (mg/kg)  Mice Alive                                                                              Mice Dead                                  ______________________________________                                        7 mg C.    14 mg non-immune                                                                             1        8                                          atrox/kg   Chick 1gG/kg                                                       7 mg C.    14 mg affinity                                                                              10        0                                          atrox/kg   purified anti-C.                                                              atrox 1gG/kg                                                       ______________________________________                                    

EXAMPLE 31 Antivenom to Notechis scutatus Venom

Notechis scutatus, an Australian elapid, produces a potent neurotoxicvenom (LD₅₀ approximately 40 μg/kg in mice). R. G. D. Theakston, In:Natural Toxins. Animal, Plant, and Microbial, (J. B. Harris, ed.)Clarendon Press, Oxford 1986, pp. 287-303. In this example, anti-N.scutatus antivenom is made according to the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. N. scutatus venom (Sigma Chemical Co. St.Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofN. scutatus venom is injected subcutaneously into three month old hensat multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of N. scutatus venom isemulsified in a 4:5 volume ratio of IFA (GIBCO) and injected intomultiple subcutaneous sites on days 14, 21, and 42.

d) Antivenom collection. Eggs, beginning on day 28, are extracted usingthe polyethylene glycol 8000 precipitation method described here inExample 1.

e) Antivenom purification. 5 mg of N. scutatus venom is coupled per mlof aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 32

Antivenom to Acanthophis antarcticus venom.

This example describes the production of a specific antivenom for A.antarcticus. This Australian elapid produces a potent neurotoxic venom.R. G. D. Theakston, In: Natural Toxins. Animal, Plant, and Microbial,(J. B. Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. Theexample comprises the following steps: a) venom detoxification, b)primary immunization, c) secondary and further immunizations, d)antivenom collection, and e) antivenom purification.

a) Venom detoxification. A. antarcticus venom (Sigma Chemical Co. St.Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofA. antarcticus venom is injected subcutaneously into three month oldhens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of A. antarcticus venomis emulsified in a 4:5 volume ratio of incomplete Freund's adjuvant(GIBCO, Grand Island, N.Y.) and injected into multiple subcutaneoussites on days 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of A. antarcticus venom is coupled perml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 33

Antivenom to Oxyuranus scutellatus venom.

In this example, production of antivenom to Oxyuranus scutellatus snakevenom is described. This Australian elapid produces a potent neurotoxicvenom (LD₅₀ approximately 150 μg/kg in mice). R. G. D. Theakston, In:Natural Toxins. Animal, Plant, and Microbial, (J. B. Harris, ed.)Clarendon Press, Oxford 1986, pp. 287-303. The example comprises thefollowing steps: a) venom detoxification, primary immunization, c)secondary and further immunizations, d) antivenom collection, and e)antivenom purification.

a) Venom detoxification. O. scutellatus venom (Sigma Chemical Co. St.Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofO. scutellatus venom is injected subcutaneously into three month oldhens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of O. scutellatus venomis emulsified in a 4:5 volume ratio of incomplete freund's adjuvant(GIBCO, Grand Island, N.Y.) and injected into multiple subcutaneoussites on days 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of O. scutellatus venom is coupled perml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 34

Antivenom to Pseudonaja textilis venom.

In this example, the production of antivenom to Pseudonaja textilissnake venom is described. This Australian elapid produces a potentneurotoxic venom. R. G. D. Theakston, In: Natural Toxins. Animal, Plant,and Microbial, (J. B. Harris, ed.) Clarendon Press, Oxford 1986, pp.287-303. The example involves the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. P. textilis venom (Sigma Chemical Co. St.Louis, MO; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofP. textilis venom is injected subcutaneously into three month old hensat multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of P. textilis venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of P. textilis venom is coupled per mlof aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 35

Antivenom to Pseudechis australis venom.

In this example the production of antivenom to Pseudechis australissnake venom is described. This Australian elapid produces a potentneurotoxic venom. R. G. D. Theakston, In: Natural Toxins. Animal, Plant,and Microbial, (J. B. Harris, ed.) Clarendon Press, Oxford 1986, pp.287-303. The example involves the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. P. australis venom (Sigma Chemical Co. St.Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofP. australis venom is injected subcutaneously into three month old hensa multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of P. australis venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of P. australis venom is coupled per mlof aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 36

Antivenom to Enhydrina schistosa venom.

In this example, the production of antivenom to Enhydrina schistosasnake venom is described. This Australian elapid produces a potentneurotoxic venom. R. G. D. Theakston, In: Natural Toxins. Animal, Plant,and Microbial, (J. B. Harris, ed.) Clarendon Press, Oxford 1986, pp.287-303. The example involves the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. E. schistosa venom (Sigma Chemical Co. St.Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah) is completelyabsorbed to bentonite by adding 1 mg of venom per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofE. schistosa venom is injected subcutaneously into three month old hensat multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of E. schistosa venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of E. schistosa venom is coupled per mlof aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. 5 ml of PEG purified crudeantivenom is applied per ml of column matrix and the unbound protein iswashed away, the specific antibody sequentially eluted and the eluentremoved, and the column regenerated as described in Example 17.

EXAMPLE 37

Antivenom to Ophiophagus hannah venom.

In this example, the production of antivenom to Ophiophagus hannah snakevenom is described. This elapid produces a potent neurotoxic venom.(LD₅₀ approximately 2.5 mg/kg in mice). R. G. D. Theakston, In: NaturalToxins. Animal, Plant, and Microbial, (J. B. Harris, ed.) ClarendonPress, Oxford 1986, pp. 287-303. The example involves the followingsteps: a) venom detoxification, b) primary immunization, c) secondaryand further immunizations, d) antivenom collection, and e) antivenompurification.

a) Venom detoxification. O. hannah venom (Sigma Chemical Co. St. Louis,Mo.; Miami Serpentarium, Salt Lake City, Utah) is completely absorbed tobentonite by adding 1 mg of venom per ml of a 2% suspension of sterilebentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofO. hannah venom is injected subcutaneously into three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of O. hannah venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of O. hannah venom is coupled per ml ofaldehyde-activated agarose matrix using the cyanoborohydride reductionmethod as described in Example 8. 5 ml of PEG purified crude antivenomis applied per ml of column matrix and the unbound protein is washedaway, the specific antibody sequentially eluted and the eluent removed,and the column regenerated as described in Example 17.

EXAMPLE 38 Antivenom to Vipera venoms

In this example, the production of antivenom to Vipera ammodytes, Viperaaspis, and Vipera berus snake venoms is described. These vipers producevenoms with strong hemorrhagic and neurotoxic properties (LD₅₀approximately 350-750 μg/kg in mice). R. G. D. Theakston, In: NaturalToxins. Animal, Plant. and Microbial, (J. B. Harris, ed.) ClarendonPress, Oxford 1986, pp.287-303. A. Ohsaka. In: Snake Venoms, (C. Y. Lee,ed.) Handbook of Experimental Pharmacology, Vol. 52, Springer Verlag,Berlin 1979, pp. 480-546. The example involves the following steps: a)venom detoxification, primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. V. ammodytes, V. berus (Sigma Chemical Co., St.Louis, Mo.; Miami (Miami Serpentarium, Salt Lake City, Utah) and V.aspis venom completely absorbed to bentonite by adding 1 mg of eachvenom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of bentonites suspension containing 1 mgof each venom (V. ammodytes, V. aspis, V. berus) is injectedsubcutaneously into three month old laying hens at multiple sites on day0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant andinjected into multiple subcutaneous sites on days 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted from egg yolks using the polyethylene glycol 8000precipitation method described herein (Example 1(c), pg. 22).

e) Antivenom purification. 5 mg of each venom is coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. The coupled matrices areblended into one column and 5 ml of PEG-purified crude antivenom isapplied per ml of column matrix. The unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 39 Antivenom to Vipera and Cerastes Venoms

In this example, the production of antivenom to Vipera xanthinapalestinae, Vipera lebetina, Cerastes cerastes, Cerastes vipera snakevenoms is described. These vipers produce venoms with strong hemorrhagicand neurotoxic properties (LD₅₀ approximately 0.5-2.0 mg/kg in mice). R.G. D. Theakston, In: Natural Toxins. Animal, Plant. and Microbial, (J.B. Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. A. Ohsaka.In: Snake Venoms. (C. Y. Lee, ed.) Handbook of ExperimentalPharmacology, Vol. 52, Springer Verlag, Berlin 1979, pp. 480-546. Theexample involves the following steps: venom detoxification, b) primaryimmunization, c) secondary and further immunizations, d) antivenomcollection, and e) antivenom purification.

a) Venom detoxification. V. x. palestinae. V. lebetina, C. cerastes(Sigma Chemical Co., St. Louis, Mo.; Miami Serpentarium, Salt Lake City,Utah) and C. vipera venom (Miami Serpentarium, Salt Lake City, Utah) iscompletely absorbed to bentonite by adding I mg of each venom per ml ofa 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofeach venom (V. x. palestinae, V. lebetina, C. cerastes, and C. vipera)is injected subcutaneously into three month old laying hens at multiplesites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant andinjected into multiple subcutaneous sites on days 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted from egg yolks using the polyethylene glycol 8000precipitation method described in Example 1.

e) Antivenom purification. 5 mg of each venom is coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. The coupled matrices areblended into one column and 5 ml of PEG-purified crude antivenom isapplied per ml of column matrix. The unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 40 Antivenom to Bitis, Vipera, and Echis Venoms

In this example, the production of antivenom to Bitis arietans, Bitisgabonica, Vipera russelli, and Echis carinatus snake venom is described.These vipers produce venoms with strong hemorrhagic and neurotoxicproperties (LD₅₀ approximately 0.25-2.0 mg/kg in mice). R. G. D.Theakston, In: Natural Toxins. Animal, Plant. and Microbial, (J. B.Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. A. Ohsaka. In:Snake Venoms, C. Y. Lee, ed.) Handbook of Experimental Pharmacology,Vol. 52, Springer Verlag, Berlin 1979, pp. 480-546. The example involvesthe following steps:

a) Venom detoxification, B. arietans, B. gabonica, V. russelli, E.carinatus (Sigma Co., St. Louis, Mo.; Miami Serpentarium, Salt LakeCity, Utah) is completely absorbed to bentonite by adding 1 mg of eachvenom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing one mlof each venom (B. arietans, B. gabonica, V. russelli, and E. carinatusis injected subcutaneously into three month old laying hens at multiplesites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant andinjected into multiple subcutaneous sites on days 14, 2I, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted from egg yolks using the polyethylene glycol 8000precipitation method described in Example 1.

e) Antivenom purification. 5 mg of each venom is coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. The coupled matrices areblended into one column and 5 ml of PEG-purified crude antivenom isapplied per ml of column matrix. The unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 41 Antivenom to Trimeresurus and Agkistrodon Venoms

In this example, the production of antivenom to Trimeresurusflavoviridis and Agkistrodon halys snake venom is described. These twosnakes inflict the most bites in Japan and their venoms contain stronghemorrhagic toxins (LD₅₀ approximately 0.8-2.7 mg/kg in mice). R. G. D.Theakston, In: Natural Toxins. Animal, Plant, and Microbial, (J. B.Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. A. Ohsaka. In:Snake Venoms, (C.-Y. Lee, ed.) Handbook of Experimental Pharmacology,Vol. 52, Springer Verlag, Berlin 1979, pp. 480-546. The example involvesthe following steps: a) venom detoxification, biprimary immunization, c)secondary and further immunizations, d) antivenom collection, and e)antivenom purification.

a) Venom detoxification. T. flavoviridis and A. halys venoms (SigmaChemical Co., St. Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah)is completely absorbed to bentonite by adding 1 mg of each venom per mlof a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of bentonite suspension containing 1 mg ofboth T. flavoviridis and A. halys venoms is injected subcutaneously intothree month old laying hens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of both T. flavoviridisand A. halys venoms is emulsified in a 4:5 volume ratio of incompleteFreund's adjuvant (GIBCO, Grand Island, N.Y.) and injected into multiplesubcutaneous sites on days 14, 21, and 42.

d) Antivenom is from eggs beginning on day 28 and shall be extractedfrom egg yolks using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom is coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. The coupled matrices areblended into a single column and 5 ml of PEG-purified crude antivenom isapplied per ml of column matrix. The unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 42 Antivenom to Naja and Hemachatus Venoms

In this example, the production of antivenom to Naja naja, Naja n. haja,Naja n. kaouthia, Naja n. oxiana, Naja n. sputatrix, Naja n. atra, Najanivea, Naja nigrocollis, and Hemachatus hemachatus snake venom isdescribed. These snakes produce potent neurotoxic venoms. R. G. D.Theakston, In: Natural Toxins. Animal, Plant and Microbial, (J. B.Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. The exampleinvolves the following steps: a) venom detoxification b) primaryimmunization, c) secondary and further immunizations, d) antivenomcollection, and e) antivenom purification.

a) Venom detoxification. N. naja, N. n. haje, N. n. kaouthia, N. n.oxiana, N. n. sputatrix, N. n. atra, N. nivea, N. nigrocollis, and H.hemachatus venoms (Sigma Chemical Co., St. Louis, Mo.; MiamiSerpentarium, Salt Lake City, Utah) are completely absorbed to bentoniteby adding 1 mg of each venom per ml of a 2% suspension of sterilebentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach venom (N. naja, N. n. haje, N. n. kaouthia, N. n. oxiana, N. n.sputatrix, N. n. atra, N. nivea, N. nigrocollis, and Hemachatushemachatus) is injected subcutaneously into three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom N. naja, N. n. haje, N. n.kaouthia, N. n. oxiana, N. n. sputatrix, N. n. atra, N. nivea, N.nigrocollis, and Hemachatus hemachatus is coupled individually per ml ofaldehyde-activated agarose matrix using the cyanoborohydride reductionmethod as described in Example 8. The coupled matrices are blended intoa single column and 5 ml of PEG-purified crude antivenom is applied perml of column matrix. The unbound protein is washed away, the specificantibody sequentially eluted, the eluent removed, and the columnregenerated as described in Example 17.

EXAMPLE 43 Antivenom to Dendroaspis Venoms

In this example, the production of antivenom to Dendroaspis angusticeps,Dendroaspis jamesonii, Dendroaspis polylepis, Dendroaspis viridis snakevenoms is described. These snakes produce potent neurotoxic venoms. R.G. D. Theakston, In: Natural Toxins. Animal, Plant and Microbial, (J. B.Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. The exampleinvolves the following steps: a) venom detoxification, b) primaryimmunization, c) secondary and further immunizations, d) antivenomcollection, and e) anti-venom purification.

a) Venom detoxification. O. angusticeps, O. jamesonii, O. polylepis andO. viridis venoms (Sigma Chemical Co., St. Louis, Mo.; MiamiSerpentarium, Salt Lake City, Utah) are completely absorbed to bentoniteby adding 1 mg of each venom per ml of a 2% suspension of sterilebentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach O. angusticeps, O. jamesonii, O. polylepis, and O. viridis venom isinjected subcutaneously into three month old hens a multiple sites onday 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom (O. angusticeps, O.jamesonii, O. polylepis and O. viridis) is coupled individually per mlof aldehyde-activated agarose matrix using the cyanoborohydridereduction method as described in Example 8. The coupled matrices areblended into a single column and 5 ml of PEG-purified crude antivenom isapplied per ml of column matrix. The unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 44 Antivenom to Bungarus Venoms

In this example, the production of antivenom to Bungarus caerulus,Bungarus fasciatus, Bungarus multicinctus snake venoms is described.These snakes produce potent neurotoxic venoms. R. G. D. Theakston, In:Natural Toxins. Animal, Plant and Microbial, (J. B. Harris, ed.)Clarendon Press, Oxford 1986, pp. 287-303. The example involves thefollowing steps: a) venom detoxification, b) primary immunization, c)secondary and further immunizations, d) antivenom collection, and e)antivenom purification.

a) Venom detoxification. B. caerulus, B. fasciatus, and B. multicinctusvenoms (Sigma Chemical Co., St. Louis, Mo.; Miami Serpentarium, SaltLake City, Utah) are completely absorbed to bentonite by adding 1 mg ofeach venom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach B. caerulus, B. fasciatus and B. multicinctus venom is injectedsubcutaneously into three month old hens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund s adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom (B. caerulus, B.fasciatus, and B. multicinctus) is coupled individually per ml ofaldehyde-activated agarose matrix using the cyanoborohydride reductionmethod as described in Example 8. The coupled matrices are blended intoa single column and 5 ml of PEG-purified crude antivenom is applied perml of column matrix. The unbound protein is washed away, the specificantibody sequentially eluted, the eluent removed, and the columnregenerated as described in Example 17.

EXAMPLE 45 Antivenom to Agkistrodon Venoms

In this example, the production of antivenom to Agkistrodon rhodostomaand Agkistrodon acutus snake venoms is described. These snakes producepotent hemorrhagic venoms. R. G. D. Theakston, In: Natural Toxins.Animal, Plant and Microbial, (J. B. Harris, ed.) Clarendon Press, Oxford1986, pp. 287-303. The example involves the following steps: a) venomdetoxification, b)primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. A. rhodostoma and A. acutus venoms (SigmaChemical Co., St. Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah)are completely absorbed to bentonite by adding 1 mg of each venom per mlof a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach A. rhodostoma and A. acutus venom is injected subcutaneously intothree month old hens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 2I, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom (a. rhodostoma and A.acutus) is coupled individually per ml of aldehyde-activated agarosematrix using the cyanoborohydride reduction method as described inExample 8. The coupled matrices are blended into a single column and 5ml of PEG-purified crude antivenom is applied per ml of column matrix.The unbound protein is washed away, the specific antibody sequentiallyeluted, the eluent removed, and the column regenerated as described inExample 17.

EXAMPLE 46 Antivenom to Bothrops and Lachesis Venoms

In this example, the production of antivenom to Bothrops atrox, Bothropsjararaca, Bothrops jararacussu, Bothrops alternatus, and Lachesis mutasnake venoms is described. These snakes produce potent hemorrhagicvenoms. R. G. D. Theakston, In: Natural Toxins. Animal, Plant andMicrobial, (J. B. Harris, ed.) Clarendon Press, Oxford 1986, pp.287-303. The example involves the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. B. atrox, B. jararaca, B. jararacussu, B.alternatus and L. muta venoms (Sigma Chemical Co., St. Louis, Mo.; MiamiSerpentarium, Salt Lake City, Utah) are completely absorbed to bentoniteby adding 1 mg of each venom per ml of a 2% suspension of sterilebentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach B. atrox, B. jararaca, B. jararacussu, B. alternatus and L. mutavenom is injected subcutaneously into>three month old hens at multiplesites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom B. atrox, B. jararaca. B.hararacussu, B. alternatus and L. muta is coupled individually per ml ofaldehyde-activated agarose matrix using the cyanoborohydride reductionmethod as described in Example 8. The coupled matrices are blended intoa single column and 5 ml of PEG-purified crude antivenom is applied perml of column matrix. The unbound protein is washed away, the specificantibody sequentially eluted, the eluent removed, and the columnregenerated as described in Example 17.

EXAMPLE 47 Antivenom to Micrurus Venoms

In this example, the production of antivenom to Micrurus corralus,Micrurus fulvius, Micrurus frontalis, and Micrurus nigrocinctus snakevenoms is described. These snakes produce potent neurotoxic venoms. R.G. D. Theakston, In: Natural Toxins. Animal, Plant and Microbial, (J. B.Harris, ed.) Clarendon Press, Oxford 1986, pp. 287-303. The exampleinvolves the following steps: a) venom detoxification, b) primaryimmunization, c) secondary and further immunizations, d) antivenomcollection, and e) antivenom purification.

a) Venom detoxification. M. fulvius and M. frontalis venoms (SigmaChemical Co., St. Louis, Mo.; Miami Serpentarium, Salt Lake City, Utah)and M. corralus and M. nigrocinctus venoms (Miami Serpentarium, SaltLake City, Utah) are completely absorbed to bentonite by adding 1 mg ofeach venom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 mg of bentonite suspension containing 1 mg ofeach M. corralus, M. fulvius, M. frontalis and M. nigrocinctus venom isinjected subcutaneously into>three month old hens at multiple sites onday 0.

c) Secondary and further immunizations. 0.25 mg of each venom above isemulsified in a 4:5 volume ratio of incomplete Freund adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of each venom M. corralus, M. fulvius,M. frontalis and M. nigrocinctus are coupled individually per ml ofaldehyde-activatedagarose matrix using the cyanoborohydride reductionmethod as described in Example 8. The coupled matrices are blended intoa single column and 5 ml of PEG-purified crude antivenom is applied perml of column matrix. The unbound protein is washed away, the specificantibody sequentially eluted, the eluent removed, and the columnregenerated as described in Example 17.

EXAMPLE 48 Production of an Avian Antivenom to a Scorpion Venom

In this example, birds were immunized with venom from a scorpion Leiurusguinguestriatus hebraeus (L.gg.). The example involveda)adsorbent/antigen mixture preparation, b) immunization, and c)antibody collection.

Adjuvant/antigen mixture preparation: L.gg. venom (Sigma) was dissolvedin distilled water and the soluble fractions used as an antigen inseveral mixtures with a 2% sterile bentonite suspension. The firstbentonite/antigen mixture consisted of 500 μg of the water solubleextract of L.cc. venom, the second bentonite antigen mixture alsoconsisted of 500 μg of the water soluble extract of L.cc. venom, thethird mixture was 400 μg of the water soluble extract of L.cc. venom. Afourth mixture was of either 0.33 mg or 0.66 mg of L.cc. venom mixedwith incomplete Freind's adjuvant in a relationship of 5:4 (adjuvant:antigen by volume) and emulsified to a firm consistency by passagethrough an antigen mixer made from two 18 gauge stainless steelhypodermic needles that had been brazed together.

b) Immunization: Two, (previously unimmunized) one-year old whiteleghorn hens (numbered for reference as #338 and #347) were immunized onday zero. Both #338 and #347 received the first adjuvant/antigen mixtureon day 0, the second adjuvant/antigen mixtures on day 14, and the thirdadjuvant/antigen mixtures on day 23. On day 57, bird #338 received 0.33mg of venom in IFA while bird #347 received 0.66 mg of venom in IFA.

c) Antibody collection: Antibody was collected from the eggs asdescribed in Example 1.

EXAMPLE 49 Covalent Attachment of the Water Soluble Toxic Fraction of aScorpion Venom to an Aldehyde-Activated Agarose Matrix

In this example, the coupling efficiency of the aldehyde activatedActigel A (Sterogene) resin (resin III) with L.cc. venom as the ligandwas demonstrated. L.gg. venom was diluted in PBS (pH 7.2) at aconcentration of 2.5 mg/ml. Resin III was washed with three volumes ofPBS and added (in equal volume) to the venom solution. Thereafter, 1/10volume of 1M sodium cyanoborohydirde (Aldrich) was added and the mixture(antigen matrix) was agitated for four hours at 4° C. The antigen matrixwas washed on a glass funnel with PBS. The filtrate was collected andcoupling efficiency was calculated as the percent of the startingprotein that was covalently attached to the matrix as described inExample 6. The results showed that the resin III coupling efficiency forL.gg. venom was 88%. The antigen matrix was stored in PBS containing0.02% sodium azide at 4° C.

EXAMPLE 50 Immunoaffinity Purification of Non-Mammalian Anti-ScorpionAntivenom from an aldehyde-Activated Scorpion Venom Antigen Matrix

In this example, the purification of scorpion venom-specific antibodieson a venom antigen matrix was demonstrated. 2.5 mg L.gg. venom wascoupled per ml of Actigel A as described in Example 49 above. Two largepools of anti-scorpion antibody were purified separately on the antigenmatrix. The first pool was 50 ml of PEG-purified antibody (8-10 mgprotein/ml; from eggs collected on day 23-32 from birds #338 and #347),the second pool was 170 ml of PEG-purified antibody (8-10 mg protein/ml;from eggs collected on days 60-68 from birds #338 and #347). Each poolwas loaded on a 2 ml scorpion venom antigen matrix at a flow rate of 1ml per minute, and washed successively with several bed volumes of PBS,BBS-Tween, and TBS until the effluent was free of protein (A₂₈₀).Anti-venom antibody was eluted with ACTISEP Elution Medium (Sterogene)with a residence time of 2 hours, and the matrix was washed with TBS.The eluate was collected and measured (A₂₈₀) after complete dialysisagainst TBS and PBS. The results showed that 0.45 mg of antibody waspurified from the first pool and 0.68 mg of antibody from the secondpool.

EXAMPLE 51 Neutralization of Scorpion Venom Lethality by an AffinityPurified Non-Mammalian Antivenom

In this example, the ability of affinity purified chicken anti-L.guinguestriatus hebraeus venom antibodies to neutralize the lethaleffects of the L.gg. venom in vivo was demonstrated. To first establishthe lethal dose of L.gg. venom in mice, water soluble extracts of venomwere diluted in saline (0.85% NaCl) to give different doses per unit ofmouse body weight. It was observed that 0.6 mg of venom per kg of bodyweight was usually fatal within 24 hours when injected intravenously.

To determine the venom neutralizing activity of the antivenom antibodiespurified as described in Example 50, the two pools of purified antibodywere combined and concentrated on a Centricon-30 concentration unit(Amicon; Bedford, Mass.) to 2.1 mg of antibody per ml of PBS. Identicalamounts of this antibody and a control non-immune chicken antibody(Cappel) were separately mixed with a fixed amount of the water solubleextract of L.egg. venom in saline, incubated for 1 hours and 100-200 μlof each mixture injected intravenously into 6-8 mice. The mice wereobserved for 24 hours and the results of this example are shown in Table10.

The statistical significance of the effect of the antivenom antibodytreated mice survival frequency was examined by Chi-square analysis andthe value for p was determined to be <0.05.

                  TABLE 10                                                        ______________________________________                                        Neutralization of Scorpion Venom In Vivo                                      Venom Dose  Antibody type                                                                              Number of Number of                                  (mg/kg)     and dose (mg/kg)                                                                           mice alive                                                                              mice dead                                  ______________________________________                                        0.63 mg L.  3.1 mg non-  1         5                                          quinquestriatus/kg                                                                        immune chicken                                                                IgG/kg                                                            0.63 mg L.  3.1 mg affinity                                                                            7         1                                          quinquestriatus 1 kg                                                                      purified anti-L.                                                              quinquestriatus/kg                                                            IgG                                                               ______________________________________                                    

EXAMPLE 52 Antivenom to Centruroides Venoms

In this example, the production of antivenom to Centruroides suffusus,Centruroides noxius, and Centruroides sculpturatus scorpion venoms isdescribed. These North American scorpions produce venoms containingseveral distinct neurotoxins. M. F. Martin et al., 1987. J. Biol. Chem.262:4452-4459. We contemplate the production of an antivenom specificfor these three scorpion venoms that comprises the following steps: a)venom detoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. C. sculpturatus (available commercially throughSigma Chemical Co., St. Louis, Mo.), C. suffusus and C. noxious venoms(obtained by milking as described by M. F. Martin et al., 1987. J. Biol.Chem. 262:4452-4459) water soluble fractions are completely absorbed byadding 0.5 mg of each venom per ml of a 2% suspension of sterilebentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of each venom is injected subcutaneously into three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of the water soluble fractions of C.sculpturatus, C. noxius, and C. suffusus venoms are coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridemethod described in Example 8 and blended into a single column. 5 ml ofPEG-purified crude antivenom is applied per ml of column matrix and theunbound protein is washed away, the specific antibody sequentiallyeluted, the eluent removed, and the column regenerated as described inExample 17.

EXAMPLE 53 Antivenom to Tityus Venom

In this example, the production of antivenom to Tityus serrulatusscorpion venoms is described. Largely a problem in South and CentralAmerica the genus Tityus includes many dangerous species including T.serrulatus (LD₅₀ approximately 1.5 mg/kg in mice). G. G. Habermehl,Venomous Animals and Their Toxins. Springer Verlag 1981, Berlin. Theexample involves the following steps: a) venom detoxification, b)primary immunization, c) secondary and further immunizations, d)antivenom collection, and e) antivenom purification.

a) Venom detoxification. T. serrulatus venom (available commerciallythrough Sigma Chemical Co., St. Louis, Mo.), water soluble fraction iscompletely absorbed by adding 0.5 mg of each venom per ml of asuspension of sterile bentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of each venom is injected subcutaneously into three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of the water soluble fraction of T.serrulatus venom is coupled per ml of aldehyde-activated agarose matrixusing the cyanoborohydride method described in Example 8. 5 ml ofPEG-purified crude antivenom is applied per ml of column matrix and theunbound protein is washed away, the specific antibody sequentiallyeluted, the eluent removed, and the column regenerated as described inExample 17.

EXAMPLE 54 Antivenom to Androctonus, Buthotus, and Buthus Venoms

In this example, the production of antivenom to Androctonus australis,Buthotus judaicus, and Buthus tamalus scorpion venoms is described.These 01d

World scorpions produce potent neurotoxic venoms (LD₅₀ 6-8 mg/kg inmice). G. G. Habermehl, Venomous Animals and Their Toxins, SpringerVerlag, 1981, Berlin. The example involves the following steps: a) venomdetoxification, b) primary immunization, c) secondary and furtherimmunizations, d) antivenom collection, and e) antivenom purification.

a) Venom detoxification. A. australis, B. judaicus, and B. tamalus(available commercially through Sigma Chemical Co., St. Louis, Mo.),water soluble venom fractions are completely absorbed by adding 0.5 mgof each venom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of each venom is injected subcutaneously into>three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of each venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBC0,Grand Island, N.Y.) and injected into multiple subcutaneous sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of the water soluble fractions of A.australis, B. judaicus, and B. tamalus venoms are coupled individuallyper ml of aldehyde-activated agarose matrix using the cyanoborohydridemethod described in Example 8 and blended into a single column. 5 ml ofPEG-purified crude antivenom is applied per ml of column matrix and theunbound protein is washed away, the specific antibody sequentiallyeluted, the eluent removed, and the column regenerated as described inExample 17.

EXAMPLE 55 Antivenom to Chironex fleckeri Venom

In this example, the production of antivenom to the jelly fish Chironexfleckeri venom is described. The box jelly fish or sea wasp, Chironexfleckeri is found in the tropical waters off Northern Australia. Thenematocysts in its tentacles contains a potent venom that can be lethalto humans (LD₅₀ in mice of crude venom approximately 0.4 mg/kg) C. E.Olson et al., 1984. Toxicon 22:733-742. The example involves thefollowing steps: a) venom detoxification, b) primary immunization, c)secondary and further immunizations, d) antivenom collection, and e)antivenom purification.

a) Venom detoxification. C. fleckeri venom is obtained from capturedspecimens as described by R. Endean, 1987. Toxicon 25:483-492. 0.5 mg ofthe crude venom will completely absorbed to each ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of each venom is injected subcutaneously into>three month old hens atmultiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of C. fleckeri venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected subcutaneously at multiple sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of C. fleckeri venom is covalentlycoupled per ml of aldehyde-activated agarose matrix using thecyanoborohydride reduction method described in Example 8. 5 ml ofPEG-purified crude anti-C. fleckeri antivenom is applied per ml ofcolumn matrix and the unbound protein is washed away, the specificantibody sequentially eluted, the eluent removed, and the columnregenerated as described in Example 17.

EXAMPLE 56 Antivenom to Black Widow Spider Venom

In this example, the production of antivenom to black widow spiderLatrodectus mantans and Latrodectus hesperus venom is described. Theblack widows (L. mactans and L. hesperus) are an especially venomousgroup of spiders (LD₅₀ approximately 1 mg/kg in mice) G. G. Habermehl.Venomous Animals and Their Toxins, Springer Verlag, Berlin 1981. Theexample involves the following steps: a) venom detoxification, b)primary immunization, c) secondary and further immunization, d)antivenom collection, and e) antivenom purification.

a) Venom detoxification. Latrodectus hesperus venom and L. m. mactansvenom saca (Sigma Chemical Co., St. Louis, Mo.) are completely absorbedto bentonite by adding 0-5 mg L. hesperus venom and the water solublefraction from 20 mg of L. mactans venom sacs per ml of a 2% suspensionof sterile bentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of each venom is injected subcutaneously into>three month old layinghens at multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of L. hesperus venom andthe water soluble fraction from 10 mg of L. mactans venom sacs areemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected subcutaneoulsy at multiple sites ondays 14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg. of L. hesperus venom and all of thewater soluble fraction from 100 mg. of L. mactans venom sacs are coupledindividually per ml of aldehyde-activated agarose matrix using thecyanoborohydride method described in Example 8 and pooled into a singlecolumn. 5 ml of PEG-purified crude antivenom is applied per ml of columnmatrix and the unbound protein is washed away, the specific antibodysequentially eluted, the eluent removed, and the column regenerated asdescribed in Example 17.

EXAMPLE 57 Antivenom to Recluse Spider Venom

In this example, the production of antivenom to Loxosceles reclusaspider venom is described. The Loxosceles genus is an important group ofvenomous group of spiders in many parts of the world. G. G. Habermehl,Venomous Animals and Their Toxins, Ch. 3, p. 22, Springer Verlag, Berlin1981. The example involves the following steps: a) venom detoxification,b) primary immunization, c) secondary and further immunizations, d)antivenom collection, and e) antivenom purification.

a) Venom detoxification. L. reclusa venom (Sigma Chemical Co., St.Louis, Mo.) is completely absorbed to bentonite by adding 0.5 mg L.reclusa venom per ml of a 2% suspension of sterile bentonite particles.

b) Primary immunization. 1 ml of a bentonite suspension containing 0.5mg of venom is injected subcutaneously into three month old laying hensat multiple sites on day 0.

c) Secondary and further immunizations. 0.25 mg of L reclusa venom isemulsified in a 4:5 volume ratio of incomplete Freund's adjuvant (GIBCO,Grand Island, N.Y.) and injected subcutaneous at multiple sites on days14, 21, and 42.

d) Antivenom collection is from eggs beginning on day 28 and shall beextracted using the polyethylene glycol 8000 precipitation methoddescribed in Example 1.

e) Antivenom purification. 5 mg of L. reclusa venom is coupled per ml ofaldehyde-activated agarose matrix using the cyanoborohydride methoddescribed in Example 8. 5 ml of PEG-purified crude antivenom is appliedper ml of column matrix and the unbound protein is washed away, thespecific antibody sequentially eluted, the eluent removed, and thecolumn regenerated as described in Example 17.

EXAMPLE 58 Assessment of Antivenom Avidity by Immunodiffusion

In this example, antivenoms to snake venoms are assessed for avidity byOuchterlony immunodiffusion gels. E. A. Kabat, Structural Concepts inImmunology and Immunochemistry (Holt, Rinehart and Winston, N.Y. 1968).A double diffusion assay is performed using the antivenoms described inExample 17. A 1% agarose solution in phosphate buffered saline (0.15MNaCl) is poured into a standard 60 mm petri dish (VWR Scientific, SanFrancisco, Calif.). After the gel hardens, a center well and sixsurrounding wells are punched out and the agarose plug removed. 10 μl ofa 100 μg/ml solution of antivenom is placed in the center well. 10 μl of100 μg/ml solutions of C. atrox and C. adamanteus venoms are placedseparately in two of the surrounding wells. The petri dish is placed at4° C. overnight and then inspected for precipitin lines.

The inspection of the petri dish revealed the presnece of precipitinlines (data not shown). This indicates that the antivenom is a highavidity antivenom.

EXAMPLE 59 In Vivo Administration of Avian Antivenom

In this example, we contemplate the treatment of humans by in vivoadministration of affinity purified antivenom following crotalidenvenomation. Treatment comprises the following steps: a) identificationof the venomous snake that has bitten the victim; b) determination ofthe severity of the envenomation; and c) intravenous administration ofpurified antivenom.

a) Identification of the venomous snake: The snake species is identifiedas a Crotalid on the basis of morphological criteria (triangular headshape, rattles, scale patterns, etc.). In view of the venomous nature ofthe offending species, the bite could involve envenomation.

b) Severity of the envenomation: clinical signs of envenomation includebut are not limited to the presence of fang marks, local swelling, pain,and necrosis at the bite site and systemic symptoms with partialparalysis, weakness, dizziness, nausea, and hemorrhage. The severity ofthe envenomation is classified on the basis of the extent of systemicsymptoms with minimal envenomation reflected by symptoms restricted tothe bite area, moderate envenomation involving tissue damage beyond thebite area, and severe envenomation characterized by tissue destructionextending beyond the bite area and strong systemic symptoms.

c) Intravenous administration of purified antivenom: Antivenom purifiedas described in Example 17 and equilibrated with phosphate bufferedsaline is sterilized by filtration through a 0.22 μm filter (Nalgene;VWR, San Francisco, Calif.), thimerosal (Sigma) added to 0.005% as apreservative, and stored in liquid form at 4° C. This sterile antivenomsolution is administered in the following dosages: 250mg for minimalenvenomation; 1000mg for moderate envenomation; and 2500 mg for severeCrotalid bites. The appropriate amount of antivenom is added to 500ml ofnormal saline for adult victims, and to 100ml of normal saline forchildren and administered intravenously using an intravenous drip line.

CONCLUSION

From the above, it is evident that the compositions and methods of thepresent invention allow for A) immunoaffinity purification of antivenomthat provides i) maximum attachment of the antigen (e.g. venom) to theresin (i.e. high attachment efficiency), ii) efficient isolation ofantigen-specific immunoglobulin that is essentially free ofnon-antigen-specific protein (i.e. the purity and quantity of antibodyobtained is optimized), iii) recovery of the antibody in an active state(i.e. the quality of reactivity is largely preserved), iv) quantitativeelution of bound antibody, v) no significant retained antibody toprogressively decrease antigen matrix capacity after successive cyclesof use (i.e. the antigen matrix is recyclable), vi) reduced burden offoreign protein in the purified antivenom, vii) retention in thepurified antivenom of the spectrum of reactivity of the unpurifiedantivenom, and viii) production of purified antivenom for treatment withreduced side effects; B) epitope determinations that provide i) means ofidentifying the monovalent and polyvalent antibody subpopulations of anexisting antivenom, ii) means of designing cost-effective immunizationcocktains for new antivenom formulas, and iii) means of designingcost-effective antigen matrices for purifying new or existingantivenoms.

With respect to retention in the purified antivenom of the spectrum ofreactivity of the unpurified antivenom, the handful of previous studiesusing whole venom describe using only single venom antigen matrices.Single venom antigen matrices are not capable of binding and purifyingthe spectrum of antivenom antibodies present in the polyvalentcommercial antivenom investigated. Thus, purification in the mannerdescribed by these researchers necessarily resulted in antivenom with amore limited reactivity than the unpurified antivenom.

In a preferred embodiment, the compositions and methods of the presentinvention allow for A) production of venom-neutralizing antivenoms inbirds which provides i) means for detoxifying venoms while preservingtheir immunogenicity, ii) means for raising large quantities ofantivenom antibodies while utilizing relatively small amounts of costlyimmunogens (i.e. the dose per unit body weight is high, but the totaldose is small), iii) means for obtaining antivenom antibodies withoutrisking injury to the animal (i.e. bleeding is not required), iv) simplemeans for obtaining whole yolk antivenom immunoglobulin that is ofgreater purity than existing horse serum antivenom ammonium sulfatefractions, and v) antivenom preparations without side-effects involvingthe host complement system.

There are several factors underlying the success of the presentinvention at obtaining the first venom-neutralizing antivenoms frombirds. Among the contributing factors are: i) the preparation ofantigens which are detoxified but immunogenic (earlier attempts atraising antivenoms to snake venom in birds were unsuccessful andinvolved glutaraldehyde-modified venom which, based upon the resultsherein described, was in all likelihood a poor immunogen), ii) the useof high doses of antigen per unit body weight (the earlier, unsuccessfulattempts at producing antivenoms to snake venom in birds may haveutilized insufficient doses of antigen to generate a broad high titerresponse to venom components), iii) purification of venom-reactiveimmunoglobulin (the earlier, unsuccessful attempts at raising antivenomto snake venom in birds utilized whole yolk immunoglobulin which did notcontain a high percentage of venom-reactive immunoglobulin, and iv)generation of high avidity antivenom (the earlier, unsuccessful attemptsat raising antivenom to snake venom in birds yielded antivenom that wasunreactive at 0.15M NaCl).

I claim:
 1. A composition comprising purified polyvalent antivenom,comprised of immunoglobulin of which greater than fifty percent isvenom-reactive, derived from a first polyvalent antivenom having two ormore monovalent subpopulations, purified such that greater than fiftypercent of said monovalent subpopulations are recovered by weight. 2.The composition of claim 1 wherein said polyvalent antivenom is in anaqueous solution in therapeutic amounts.
 3. The composition of claim 2wherein said polyvalent antivenom is intravenously injectable.
 4. Thecomposition of claim 1 wherein said purified antivenom reacts with twoor more venoms selected from the group consisting of Bitis arietans,Bitis gabonica, Vipera russelli, Echis carinatus, Trimeresurusflavoviridis, and Agkistrodon halys.
 5. The composition of claim 1wherein said polyvalent antivenom comprises horse antibody.
 6. Thecomposition of claim 1, wherein said polyvalent antivenom is derivedfrom a first polyvalent antivenom comprised of immunoglobulin of whichless than fifty percent is venom-reactive, and has substantially thesame spectrum of reactivity as said first polyvalent antivenom.
 7. Acomposition comprising, polyvalent antiveno, derived from a firstpolyvalent antivenom comprised of immunoglobulin of which less thanfifty percent is venom-reactive, purified such that greater than fiftypercent of said immunoglobulin is venom-reactive and such that saidpurified antivenom has substantially the same spectrum of reactivity assaid first polyvalent antivenom.
 8. The composition of claim 7 whereinsaid polyvalent antivenom is in an aqueous solution in therapeuticamounts.
 9. The composition of claim 7 wherein said polyvalent antivenomis intravenously injectable.
 10. The composition of claim 7 wherein saidpurified polyvalent polyvalent antivenom reacts with two or more venomsselected from the group consisting of Bitis arietans, Bitis gabonica,Vipera russelli, Echis carinatus, Trimeresurus flavoviridis, andAgkistrodon halys.
 11. The composition of claim 7 wherein saidpolyvalent antivenom comprises horse antibody.
 12. The composition ofclaim 7 wherein said polyvalent antivenom comprises two or moremonovalent subpopulations.
 13. The composition of claim 1 wherein saidpolyvalent antivenom comprises antibody with reactivity to C. atrox, B.atrox, C. adamanteus, and C. durissus terrificus venom.
 14. Thecomposition of claim 13 wherein one of said monovalent subpopulationscomprises antibody with reactivity to C. durissus terrificus venom. 15.The composition of claim 7 wherein said polyvalent antivenom comprisesantibody with reactivity to C. atrox, B. atrox, C. adamanteus, and C.durissus terrificus venom.
 16. The composition of claim 15 wherein saidpolyvalent antivenom further comprises one monovalent subpopulation withreactivity to C. durissus terrificus venom.
 17. A method of producingvenom-neutralizing antivenom, comprising:a) providing a plurality ofimmunogenic immunizing venoms; b) providing a plurality of purifyingvenoms selected from among said immunizing venoms; c) providing at leastone host species d) immunizing said host species with a plurality ofimmunizing venoms, so that a first neutralizing polyvalent antivenomhaving two or more monovalent subpopulations is produced; and e)purifying said first polyvalent antivenom by contacting said firstpolyvalent antivenom sequentially or simultaneously with said purifyingvenoms, so that a purified second neutralizing polyvalent antivenom isproduced wherein said second neutralizing polyvalent antivenom iscomprised of immunoglobulin of which greater than fifty percent is venomreactive and purified such that greater than fifty percent of saidmonovalent subpopulations are recovered by weight.
 18. A method ofproducing venom-neutralizing antivenom, comprising:a) providing aplurality of immunogenic immunizing venoms; b) providing purifyingvenoms consisting of fewer than all of the immunizing venoms; c)providing at least one host species; d) immunizing said host specieswith a plurality of immunizing venoms, so that a first polyvalentantivenom is produced; and e) purifying said first polyvalent antivenomusing said purifying venoms, so that a purified second polyvalentantivenom is produced having substantially the same spectrum ofreactivity as said first polyvalent antivenom.
 19. A method of producingvenom-neutralizing antivenom, comprising:a) providing C. durissusterrificus venom and at least one additional venom in an immunogenicmixture; b) providing at least one host species; c) immunizing said hostspecies with said venom mixture, so that a first polyvalent antivenomhaving two or more monovalent subpopulations is produced; and d)purifying said first polyvalent antivenom, comprising contacting saidfirst polyvalent antivenom with C. durissus terrificus venom, so that apurified second neutralizing polyvalent antivenom is produced whereinsaid second neutralizing polyvalent antivenom is comprised ofimmunoglobulin of which greater than fifty percent is venom reactive andpurified such that greater than fifty percent of said monovalentsubpopulations are recovered by weight.
 20. A composition comprisingvenom-neutralizing avian IgY antivenom.
 21. The composition of claim 20wherein said avian antivenom is chicken antivenom.
 22. The compositionof claim 20 wherein said avian antivenom is comprised of proteincomprised of greater than 90% immunoglobulin.
 23. The composition ofclaim 20 wherein said avian antivenom comprised of protein comprised ofgreater than 99% immunoglobulin.
 24. The composition of claim 20 whereinsaid avian antivenom is comprised of protein comprised of greater than50% venom-reactive immunoglobulin.
 25. The composition of claim 20wherein said antivenom is in an aqueous solution in therapeutic amounts.26. The composition of claim 25 wherein said antivenom is intravenouslyinjectable.
 27. The composition of claim 20 wherein said avian antivenomreacts with venom selected from the group consisting of Bitis arietans,Bitis gabonica, Vipera russelli, Echis carinatus, Trimeresurusflavoviridis, C. atrox, B. atrox, C. adamanteus, C. durissus terrificus,andAgkistrodon halys.
 28. The composition of claim 20 wherein said avianantivenom is polyvalent.
 29. A method of producing venom-neutralizingantivenom, comprising:a) providing one or more immunogenic immunizingvenoms, each comprising one or more components responsible for toxicity;b) providing at least one avian species; c) reducing said toxicity ofsaid components without rendering said one or more venomsnon-immunogenic; d) immunizing said avian species with said one or moreimmunizing venoms, so that a neutralizing antivenom is produced in theeggs of said avian species; and e) isolating said antivenom from saideggs.
 30. The method of claim 29 wherein said immunizing venoms aresnake venoms selected from the group consisting of Bitis arietans, Bitisgabonica, Vipera russelli, Echis carinatus, Trimeresurus flavoviridis,C. atrox, B. atrox, C. adamanteus, C. durissus terrificus, andAgkistrodon halys.
 31. The method of claim 29 wherein said avian speciescomprises chickens.